The nuclear matrix is involved in the replicative cycle of herpes simplex virus type 1 (HSV-1) and in at least some cases viral DNA has been shown to be closely associated with this structure. In this communication, we report the presence of five DNA-binding proteins in the nuclear matrix of HSV-1-infected BHK cells. These proteins (p114, p89, p77, p37 and p29) were detected by probing with 32P-labelled HSV DNA after Western blotting of nuclear matrix proteins. Three were identified as virion components: p89 as VP12, p77 as VP13 and p37 as the capsid protein VP22a. These polypeptides were detected in cells and nuclei and found to be associated with the nuclear matrix late during the lytic cycle, long after the onset of viral DNA replication. The nuclear matrix-binding capacity of VP22a depended on viral DNA replication, since after DNA polymerase inhibition it was still synthesized and transported into the nucleus but was no longer associated with the nuclear matrix. After inhibition of viral DNA synthesis, VP13 was no longer found in cells, nuclei or nuclear matrices. These results suggest a possible involvement in anchoring viral progeny DNA to the nuclear matrix.