Bovine parvovirus DNA is associated with the nuclear matrix of infected bovine fetal lung cells as shown by Southern blot analysis of matrix DNA isolated by two procedures differing in the order of exposure of detergent-treated nuclei to high salt conditions and DNase I. Protein analysis of the two matrix types showed the polypeptide composition to be similar. Both procedures showed enrichment for BPV DNA with progressive DNase I digestion. Over the course of infection the amount of BPV DNA associated with the matrix increased, yet the amount of BPV DNA associated with matrix DNA as opposed to total DNA decreased from 21% at two hours to 7% at eight hours with a subsequent rise to 13% at sixteen hours. Restriction enzyme analysis of the matrix DNA indicated that no specific portion of the BPV genome was responsible for its attachment to the matrix at the selected times. In addition both the nonstructural BPV protein, NP-1, and the capsid proteins VP1, VP2, and VP3 were associated with the matrix at sixteen hours. The association of BPV DNA and proteins with the nuclear matrix implies structural if not functional significance for the matrix in BPV replication.