The phenotypes of cultured cell lines established from individuals with Bloom syndrome (BLM), including an elevated spontaneous frequency of sister chromatid exchanges (SCEs), are consistent with a defect in DNA joining. We have investigated the levels of DNA ligase I and DNA ligase III in an SV40-transformed control and BLM fibroblast cell line, as well as clonal derivatives of the BLM cell line complemented or not for the elevated SCE phenotype. No differences in either DNA ligase I or DNA ligase III were detected in extracts from these cell lines. Furthermore, the data indicate that in dividing cultures of SV40-transformed fibroblasts, DNA ligase III contributes > 85% of high molecular weight DNA joining activity. This observation contrasts with previous studies in which DNA ligase I was reported to be the major DNA joining activity in extracts from proliferating mammalian cells.