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Topoisomerase I has ubiquitous roles in important cellular functions such as replication, transcription, and recombination. In order to further characterize this enzyme in vivo, we have used gene targeting to inactivate the mouse Top-1 gene. A selection protocol using the topoisomerase I inhibitor camptothecin facilitated isolation of embryonic stem cell clones containing an inactivated allele; isolation of correctly targeted clones was enhanced 75-fold over that achieved by normal selection procedures. The disrupted Top-1 allele is embryonic lethal when homozygous, and development of such embryos fails between the 4- and 16-cell stages. Both sperm and oocytes containing the inactive allele maintain viability through the fertilization point, and thus gene expression of topoisomerase I is not required for gamete viability. These studies demonstrate that topoisomerase I is essential for cell growth and division in vivo. The Top-1 gene was also shown to be linked to the agouti locus.