Tenebrio molitor eggs were sterilized with zephiran chloride, hatched, and the larvae and adults reared under germfree conditions. The beetle larvae were infected with surface sterilized Hymenolepis diminuta oncospheres. Cysticercoids recovered from the infected larvae were fed to germfree and conventional rats. A second group of conventional rats were infected with conventional cysticercoids. Germfree and conventional control rats were autopsied at varying intervals after infection. Adult worms bearing gravid proglottids were recovered, completing the parasite life cycle under germfree conditions. Lengths and wet weights of worms were recorded and viability of oncospheres was determined by hatching in vitro and by feeding to conventional Tribolium confusum and subsequently recovering cysticercoids. Tapeworms were stained and examined morphologically. No differences were detected between adult or larval tapeworms from germfree and conventional hosts. Studies of animal parasites in germfree hosts are still relatively few. Most experiments have been concerned with nematode and protozoan parasites and the results of these studies have been diverse. Workers have found that certain parasites are unable to grow in the absence of microorganisms, grow less successfully, or are less pathogenic when in germfree hosts (Phillips, 1964; Phillips et al., 1958; Doll and Franker, 1963; Wescott and Todd, 1964). Some parasites develop normally in both germfree and conventional hosts (Chernin, 1960; Griesemer et al., 1963), whereas still others that fail to develop in a given conventional host will do so when that host is germfree (Newton et al., 1959). Hymenolepis diminuta was selected for this study because of the ready availability of both parasites and hosts, the rat and the flour beetle, Tenebrio molitor. In contrast to most of the parasites previously studied in germfree hosts, H. diminuta has both a definitive and an intermediate host, and the effects of the lack of a normal flora of either host on the development of the cestode could be examined. Furthermore, it seemed likely that due to its absorption method of nutrition, a cestode might have a need for some of the metabolic by-products of the host gut flora. A study of the influence of microorganisms on the development of H. diminuta was made by deReceived for publication 13 September 1967. * This study supported by the University of Wisconsin Graduate School. Project No. 67-13080-1. termining if it was possible for the parasite to reach maturity in germfree hosts. MATERIALS AND METHODS Germfree animals in all experiments were maintained in a 24by 60-inch Sta-Safe flexible-film gnotobiotic chamber (Standard Safety Equipment Co. No. SD4205-56). Establishment of germfree beetle colonies Tenebrio molitor eggs were removed from stock beetle cultures and surface sterilized by immersion for 3 min in a solution of 1:3,000 zephiran chloride in distilled water. Receiving dishes for the sterilized eggs were prepared by lining the bottom of 10-cm petri dishes with a double layer of Reeve Angel Grade 202 filter paper. Ten 1/4-inch squares of filter paper were spaced on the layer and the dishes then covered and autoclaved at 15 lb pressure for 15 min. The zephiran chloride-treated eggs were taken up one at a time with a sterile pipette and placed on a filter paper square in the receiving dishes. After excess solution had drained from the eggs, each square with its egg was aseptically transferred to a nutrient agar plate. The agar surface had previously been covered with a 9-cm disc of autoclaved filter paper (RA Grade 202). As each egg hatched, the larva was transferred to a petri dish containing sterile chicken feed or stone ground flour. After 2 months, each larva and a small quantity of food from the petri dish in which it had been kept were tested for sterility on nutrient and blood agar culture plates. If no growth of microorganisms was observed after 2 weeks' incubation, the tested larva was placed in a sterile, screw-cap vial and introduced into the germfree chamber. Inside the chamber, the larvae were kept in 250-ml, cotton-stoppered, Erlenmeyer flasks containing approximately 40 g of chicken feed. The chicken feed used in all germfree beetle cultures was a special formula recommended by the University of Wisconsin Depart-