Brefeldin A (BFA) was found to interfere with specific events of human cytomegalovirus (HCMV) maturation in human fibroblasts. Ultrastructural as well as biochemical studies suggested that short-term exposure of infected cultures to BFA during the late infectious cycle primarily prevented Golgi-dependent processes, e.g. envelopment of naked cytoplasmic nucleocapsids in the trans-Golgi network (TGN) and normal processing of glycoprotein B. In contrast, the nuclear phase of viral morphogenesis, e.g. transport budding at the nuclear envelope, was not impaired. These observations were compatible with the interpretation that HCMV morphogenesis may involve sequential budding events at the nuclear envelope and at cisternae of the TGN. BFA treatment during the early infectious cycle efficiently inhibited HCMV-DNA synthesis and thus late viral functions, preventing production of viral progeny. Cytotoxicity was excluded as a cause for these findings.