Stages in the life cycle of Eimeria tenella grown in cell culture were investigated for cytochemical changes during development and the results were compared with previous in vivo studies. Chromatin rings composed of DNA were present in all stages from sporozoite to microgametocyte but were absent in macrogametocytes. Nuclear RNA occurred in the nucleoli of sporozoites and macrogametocytes. The cytoplasm of all schizonts reacted strongly for RNA while some nuclei contained positive acentric granules which were retained in the nuclei of merozoites. Carbohydrates and lipids were found in greater quantities than were described from studies based on in vivo preparations. Though carbohydrates disappeared during early sporozoite development, they accumulated during schizogony and gametogenesis. Lipid droplets occurred in all life cycle stages and were particularly concentrated within residual or unsegmented areas of schizonts and microgametocytes. Acid mucopolysaccharides, detected only after alcoholic fixation, were concentrated in cytoplasmic granules found in all developmental forms of the parasite. Tyzzer's early work on coccidian development (1929) was the basis of later cytochemical observations (Gill and Ray, 1954a, b; Pattillo and Becker, 1955; Cheissin, 1958, 1960). Recent studies on the ultrastructure of coccidia have shown numerous inclusions that were not explained by previous workers, thus pointing out the need for more precise cytochemical approaches. Fayer and Hammond (1967), Doran (1970), and Strout and Ouellette (1970) showed that cell culture provides an excellent opportunity to observe coccidian development as embedding procedures, sectioning, and variations in host cell type are eliminated. Transition stages are readily observed and development can be followed in sequence from sporozoite to gamete. We attempted to determine if cell culture could provide additional metabolic information and to see if cytochemical variations exist between in vivo and in vitro development. MATERIALS AND METHODS We fixed and stained monolayers of chick embryo kidney cells, grown on cover slips in Received for publication 6 July 1971. * From a thesis submitted in partial fulfillment of the requirements for the degree of Master of Science, Department of Zoology. Supported in part by Research Grant AI 05745-06 from the NIAID, U. S. Public Health Service, and by a grant-in-aid from Eli Lilly Co., Greenfield, Ind. Published with the approval of the director of the New Hampshire Agricultural Experiment Station as Scientific Contribution No. 483. Leighton tubes and infected with 200,000 sporozoites as described by Strout and Ouellette (1970) and Strout, Ouellette, and Gangi (1969a, b). We withdrew cover slips 3, 6, and 24 hr after inoculation and at 24-hr intervals thereafter to 216 hr. The use of monolayers for cytochemical procedures permitted rapid penetration of stains and fixatives and allowed us to stain the cover slips in petri dishes and to take tlie cultures directly from formalin fixatives to water. For each cytochemical procedure we stained noninfected control and infected cultures concurrently. To determine the extent of parasite development at each withdrawal period, we also used Giemsa's stain on infected cultures. For each chemical property studied, we tried a variety of stains and fixatives; those combinations which repeatedly gave the best results are as follows: For the study of proteins and carbohydrates we fixed monolayers in neutral buffered 10% formalin (Lillie, 1954) or Carnoy's fluid. Proteins, identified with the bromphenol blue method of Mazia, Brewer, and Alfert (1953), modified by omitting the alkaline wash, were confirmed by peptic digestion (0.1 mg/ml in 0.1 N HC1). To identify carbohydrates we stained the cultures by the periodic acid-Schiff (PAS) method of Thompson (1966) using 5 min oxidation in 0.5% periodic acid. We tested the specificity of the PAS method by acetylation in alcohol-ammonium hydroxide at 37 C (Lillie, 1954). Because we identified a glycogenlike polysaccharide as being labile after saliva digestion, we confirmed this observation by using the chromic acid-Schiff reaction (Bauer, 1933). After fixation in Carnoy's fluid acid mucopolysaccharides with sulfated esters (which remained stable after incubation in bovine testis hyaluronidase, 1 mg/ml in phosphate buffer pH 7.0) were detected by the alcian blue method (Steedman, 1950) and confirmed by toluidine blue metachromasia (Pearse, 1960). We