Intranuclear redistributions of DNA polymerase alpha activity (the presumptive replicative enzyme) are observed preceding and during in vivo replication in regenerating rat liver. The most marked induction of in vitro enzyme activity occurs coincident with the onset of in vivo replication and is seen in the high salt resistant domain of the nuclear matrix. Moreover, DNA polymerase alpha endogenous and exogenous template activities begin to redistribute prereplicatively from bulk chromatin directly to the high salt resistant matrix. These prereplicative subnuclear events are detectable prior to the induction of total cellular DNA polymerase alpha and appear to continue throughout replication. Maximal percent recovery of the total nuclear activity on the high salt resistant matrix (approximately 30%) occurs coincident with and during the time when in vivo replication rates are maximum. At the conclusion of replication, DNA polymerase alpha activities shift back into the bulk chromatin and are no longer high salt resistant. In contrast, DNA polymerase beta (presumptive repair enzyme) endogenous and exogenous template activities are randomly distributed in the nuclear subfractions through the proliferative response. We conclude that DNA polymerase alpha is dynamically assembled or activated on the nuclear matrix and that this process is initiated before the onset of in vivo replication.