DNA topoisomerase I from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe was overproduced using the cloned genes. Extracts from cells overproducing DNA topoisomerase I were prepared and incubated with 32P-labeled DNA. Alkali was used to trap the topoisomerase I-DNA covalent intermediate. Most of the DNA was digested with nuclease, and the resultant 32P-labeled topoisomerase I was subjected to cleavage with cyanogen bromide or formic acid. From the molecular weights of the resultant labeled peptides and by comparison of the amino acid sequences derived from the cloned genes, we were able to deduce that the active site tyrosine of eukaryotic DNA topoisomerase I is very near the carboxyl terminus, at amino acid 771 for S. pombe and 727 for S. cerevisiae. Site-directed mutagenesis was used to change tyrosine 727 of S. cerevisiae topoisomerase I to a phenylalanine. The resulting mutant topoisomerase I protein lost all DNA relaxation activity and rendered cells resistant to the topoisomerase I inhibitor, camptothecin. The amino acid sequence of human topoisomerase I has significant similarity to the two yeast topoisomerase I sequences. Based on this similarity, we infer that tyrosine 723 is the active site tyrosine of human enzyme.