The effect of Mycoplasma pulmonis suspension on mouse on mouse fertilization and preimplantation development in vitro was examined. When sperm were preincubated with M. pulmonis, fertilization of eggs occured less consistently than when untreated sperm were used. There was also a highly significant and consistent reduction in embryonic develoment in the treated group, with relatively few embryos reaching the blastocyst stage. The adverse effects were not seen with M. fermentans or with M. pulmonis organisms inactivated by heating or sonication before incubation with gametes. Likewise, preincubation of M. pulmonis in medium containing tetracycline, an antibiotic which interferes with protein synthesis, resulted in embryonic development similar to that seen in the untreated group. These results suggest that the deleterious effects obtained with viable mycoplasmas are due to some substance produced by their active metabolism. Other results indicate that the egg is susceptible to M. pulmonis for a limited time, since incubation of two-cell embryos with mycoplasmas had no effect on development to the blastocyst stage. The findings are discussed in relation to the possible association between human infertility and mycoplasma infection.