The immediate-early (IE) genes of human cytomegalovirus (CMV) can be expressed in monocytic cells and are known to regulate viral and cellular genes. Reactivation of human immunodeficiency virus (HIV-1) may be stimulated by a variety of factors including other viruses and inflammatory cytokines. These studies examine the role of hyperthermia and CMV in the regulation of HIV-1 and tumor necrosis factor (TNF)-α. THP-1 cells were transfected with the CMV IE genes. HIV-1 and TNF-α transcription were assessed with chloramphenicol acetyltransferase promoter constructs. Hyperthermia sufficient to stimulate production of heat shock proteins was used to stimulate the cells. Hyperthermia significantly enhances the effect of CMV IE gene products on the expression of HIV-1 and TNF-α. The increases in HIV-1 transcription appear to be in part due to increases in TNF-α. Heat shock proteins induced by hyperthermia may play an important role in the viral regulation of monocytic function by CMV.