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To directly assess c-myc function in cellular proliferation, differentiation, and embryogenesis, we have used homologous recombination in embryonic stem cells to generate both heterozygous and homozygous c-myc mutant ES cell lines. The mutation is a null allele at the protein level. Mouse chimeras from seven heterozygous cell lines transmitted the mutant allele to their offspring. The analysis of embryos from two clones has shown that the mutation is lethal in homozygotes between 9.5 and 10.5 days of gestation. The embryos are generally smaller and retarded in development compared with their littermates. Pathologic abnormalities include the heart, pericardium, neural tube, and delay or failure in turning of the embryo. Heterozygous females have reduced fertility owing to embryonic resorption before 9.5 days of gestation in 14% of implanted embryos. c-Myc protein is necessary for embryonic survival beyond 10.5 days of gestation; however, it appears to be dispensable for cell division both in ES cell lines and in the embryo before that time.