Monocyte-derived macrophages were co-infected with human immunodeficiency virus (HIV), strain HIVBa-L, and human cytomegalovirus (HCMV) strain AD 169. HCMV enhanced the production of p24 antigen and infectious HIV, but HIV had no effect on HCMV production. In dually infected cultures HIV p24 antigen levels were increased 5- to 15-fold during the first 3 weeks of culture. This enhancement was observed both for cell-associated and extracellular p24 antigen from co-infected cells. The replication of macrophage-tropic clinical isolates of HIV were also enhanced by HCMV, but HCMV did not enable T cell-tropic viruses to replicate in macrophages. Conversely, HCMV clinical isolates from blood or urine were able to upregulate HIV production. Similar enhancement of HIV p24 antigen levels was observed with noninfectious, heat-inactivated HCMV, indicating that replication of HCMV in the macrophages was not necessary. Although replication was not required, the enhancing ability of HCMV was associated with the virus. Enhancement of HIV segregated with the HCMV pelleted virus particles and not the supernatant in stock virus. The fact that HCMV and HIV are detected in macrophages in persons with HIV infection highlights the importance of this system for studying interactions between HIV and HCMV in persons with HIV infection.