Bovine DNA ligases I and II were adenylylated in the presence of [alpha-32P]ATP and digested with limiting amounts of trypsin or V8 protease. The generation of radioactive peptides of decreasing size was monitored by polyacrylamide gel electrophoresis and autoradiography. Active site peptides obtained by complete proteolytic digestions with trypsin, V8, or Lys-C protease were also compared. The partial digestion products of DNA ligases I and II were entirely different, with no indication of extensive sequence homology. Furthermore, the sequence of the active site region of DNA ligase I is clearly different from that of DNA ligase II. Similar analysis of a third chromatographically distinct mammalian DNA ligase indicated that it is different from DNA ligase I but related to DNA ligase II.