Although DNA polymerase-alpha (DNA nucleotidyltransferase; deoxynucleoside triphosphate: DNA deoxynucleotidyltransferase; EC 2.7.7.7) probably functions in the nucleus, it is usually found predominantly in the nonnuclear fraction of disrupted cells. We have reexamined the intracellular location of this enzyme using cytochalasin-B-induced enucleation, a technique which avoids exposure of nuclei to extra-cellular conditions during cell fractionation. In conditions where viability of separated cell parts is high and recovery is quantitative, we find greater than 85% of total DNA polymerase-alpha (and DNA polymerase-beta) activity in the nucleated cell fragments (karyoplasts), from which we conclude that the location in vivo of DNA polymerase-alpha is either nuclear or perinuclear. On the other hand, thymidine kinase (ATP: thymidine 5'-phosphotransferase, EC 5.7.1.75) is found primarily in the enucleated cell fragments (cytoplasts). The enucleation procedure used in this work should be of general use for intracellular location studies.