Experiments were undertaken to identify the product of open reading frame (orf) UL56 of human cytomegalovirus (HCMV), the putative homolog of infected cell protein (ICP) 18.5 of herpes simplex virus type 1 (HSV-1) that is thought to be involved in viral nucleocapsid maturation. Northern blotting using a unique fragment of orf UL56 revealed a specific transcript of about 3.0 kb in HCMV-infected fibroblasts early and late postinfection (p.i.). Two overlapping fragments of UL56 were subcloned for procaryotic expression and the recombinant proteins were used for affinity purification of an antibody fraction from human convalescent serum (pab UL56). The affinity purified antibody recognized a polypeptide with an apparent molecular weight of about 130 kDa (p 130) in immunoblots of virions and of infected cell extracts late p.i. Upon treatment of isolated virions with nonionic detergent the p 130 polypeptide separated with the nucleocapsid/tegument fraction. The amino acid sequence of p 130 deduced from the nucleotide sequence exhibited a high homology to ICP 18.5 of HSV-1.