Genetic studies have previously demonstrated that the Saccharomyces cerevisiae CDC9 gene product, which is functionally homologous to mammalian DNA ligase I, is required for DNA replication and is also involved in DNA repair and genetic recombination. In the present study we have purified the yeast enzyme. When measured under denaturing conditions, Cdc9 protein has a polypeptide molecular mass of 87 kDa. The native form of the enzyme is an 80-kDa asymmetric monomer. Both estimates are in good agreement with the M(r) = 84,406 predicted from the translated sequence of the CDC9 gene. Cdc9 DNA ligase acts via the same basic reaction mechanism employed by all known ATP-dependent DNA ligases. The catalytic functions reside in a 70-kDa C-terminal domain that is conserved in mammalian DNA ligase I and in Cdc17 DNA ligase from Schizosaccharomyces pombe. The ATP analog ATP alpha S inhibits the ligation reaction, although Cdc9 protein does form an enzyme-thioadenylate intermediate. Since Cdc9 DNA ligase exhibited the same substrate specificity as mammalian DNA ligase I, this enzyme can be considered to be the DNA ligase I of S. cerevisiae. There is genetic evidence suggesting that DNA ligase may be directly involved in error-prone DNA repair. We examined the ability of Cdc9 DNA ligase to join nicks with mismatches at the termini. Mismatches at the 5' termini of nicks had very little effect on ligation, whereas mismatches opposite a purine at 3' termini inhibited DNA ligation. The joining of DNA molecules with mismatched termini by DNA ligase may be responsible for the generation of mutations.