A nuclear matrix-bound in vitro replicational system that utilizes alpha-polymerase and matrix-attached DNA has been isolated from regenerating rat liver and characterized for in vitro requirements. beta-Polymerase is a minor component of the matrix in vitro system. Evidence is presented for the native association of alpha-polymerase with the nuclear matrix and for the parallel induction of matrix-bound alpha-polymerase and in vivo DNA synthesis during the proliferative response following partial hepatectomy. Our data suggest that the attachment of alpha-polymerase to the matrix may be an important step in the assembly of functional replisomes. We also demonstrate prereplicative and late-replicative rises in nuclear and matrix-bound beta-polymerase activity and discuss these results in terms of possible genome screening.