The biochemical defect in Bloom's syndrome (BS) remains unknown, but two characteristic features of BS cells point to a disturbance of DNA replication, namely, an excessive number of sister-chromatid exchanges (SCEs) in bromodeoxyuridine (BrdU)-substituted cells and an abnormally slow rate of replicon elongation. The hypothesis of an abnormal DNA polymerase as the explanation for these observations was tested using an in situ assay system for DNA polymerase activity and to estimate molecular weights in cellular extracts of cultured BS cells. DNA polymerase subunits in cellular extracts from the BS cells when separated electrophoretically on polyacrylamide gels showed the same mobilities (i.e., molecular weights) as the controls and were equally effective at promoting the incorporation of isotopically labeled nucleosides. It is concluded that the genetic defect in BS has no direct effect on either DNA-polymerase activity or the amounts and molecular weights of the different forms of the enzyme.