The activity of 2 nonmitochondrial forms of DNA polymerase, designated DNA polymerases alpha and beta, was investigated during liver regeneration in regimented rats. In accord with Barbiroli and Potter, we observed that regimentation of rats with respect to temperature, light and darkness, and availability of food resolves the DNA synthesis response to partial hepatectomy into 2 peaks, one occurring at a fixed time after operation and the other entrained by the environmental conditions. The peaks can be fused or separated depending on the timing of the operation. For this study, operation times were selected to give both patterns of DNA synthesis as measured by the uptake of radioactive thymidine into DNA. For both operation times, DNA polymerase activity in the nuclear extract correlated temporally and qualitatively with radioactive thymidine uptake into DNA. At the times of maximal DNA synthesis and polymerase activity, the DNA polymerase was purified from extracts of isolated nuclei. DNA polymerase alpha represented 70% and DNA polymerase beta represented 30% of the recovered activity from the nuclear extract. This is in agreement with the previous observation in nonregimented rats that DNA polymerase alpha is the major activity in nuclei during liver regeneration. For both operation times, DNA polymerase activity in the postmicrosomal fraction was sedimentable and increased 3 to 4 times above the level observed with this same fraction from normal rat liver. This activity was shown to be due to DNA polymerase alpha only with this subcellular fraction. DNA polymerase alpha activity with this fraction peaked 4 to 6 hr after the time of maximal radioactive thymidine incorporation into DNA. DNA polymerase activity in the microsome fraction did not change significantly after partial hepatectomy. This activity has been shown to represent DNA polymerase beta. Prior administration of cycloheximide and actinomycin abolished the rise in DNA polymerase alpha activity in the nucleus and postmicrosomal fraction. Hydroxyurea did not prevent the rise in DNA polymerase alpha activity with those subcellular fractions but did inhibit over 90% of the uptake of radioactive thymidine into DNA. These data suggest, but do not prove, that DNA polymerase alpha activity is induced in response to the stimulus(i) for liver regeneration.