An understanding of the physiological role of a particular enzyme in cell metabolism rests in part on a knowledge of the exact intracellular site of action of that enzyme. Both histochemical tests (22) and the differential centrifugation of cell free homogenates (10) have yielded valuable information concerning the intracellular location of some enzymes in plants. Unfortunately, reliable histochemical tests are available for only a few enzymes, and the possibility of artifacts arising as a result of isolation procedures often negates the conclusions obtained by means of the differential centrifugation technique. Of particular importance in this connection are the leaching of enzymes from morphological units and the possibility of adsorption of soluble enzymes by such structures during their isolation in aqueous media. A further difficulty that may be encountered is the inactivation of enzymes resulting from a mixing of the cell contents during grinding and separation in aqueous media (7). The extensive use, in physiological and biochemical studies, of chloroplast isolation in aqueous media makes it imperative that we increase our knowledge concerning the effects of these techniques on the loss of large molecular weight substances such as enzymes and cofactors from the plastids during their isolation. This point has been emphasized by McClendon (14) in his studies on the effect of polyethylene glycol upon the loss of phycocyanin from red algal chloroplasts. The necessity of adding a cytoplasmic back to isolated chloroplasts to attain maximum activity is indicative of the problem. A water soluble chloroplast extract appears to function in phosphorylation by reducing triphosphopyridine nucleotide in the light (4, 17). Similarly Avron and Jagendorf (5) found an extractable chloroplast factor associated with photosynthetic phosphorylation. Certain water soluble enzymes, which because of their role in photosynthesis or in starch synthesis would be expected to be found chiefly in chloroplasts, have been reported to be present primarily in the supernatant from chloroplasts isolated in aqueous media. This is true of aldolase (21) and phosphorylase (15,20). In order to overcome some of the difficulties involved in the isolation of nuclei in aqueous media, Behrens (6) developed a procedure whereby nuclei could be isolated from lyophilized heart muscle without the use of aqueous solutions. This method has been modified recently and used in the study of nuclei isolated from animal tissue (1, 8, 9, 12). However, with the exception of the isolation of wheat germ nuclei (19), the nonaqueous technique has not been reported to have been employed on plant material.