Because mycoplasma infection has often been observed in individuals infected with human immunodeficiency virus type 1 (HIV-1), we investigated the influence of mycoplasmas on HIV replication in vitro. Replication of HIV-1 assessed by reverse transcriptase activity and the p24 antigen level in peripheral blood mononuclear cells (PBMCs) was enhanced by the stimulation with the cell lysates of human mycoplasmas such as Mycoplasma pneumoniae, M. genitalium, and M. fermentans. The most drastic increase of HIV-1 replication was shown in M. pneumoniae, which induced an approximately 10-fold higher replication level than phytohemagglutinin. Stimulation with the mycoplasmas accompanied the induction of blastotransformation of CD4+ but not CD8+ T lymphocytes in PBMCs and the most predominant alterations were induced by the stimulation with M. pneumoniae among the species examined. Production of IL-2 was significantly increased by stimulation with all three species of Mycoplasma used. These results suggest that mycoplasmas induce vigorous replication of HIV-1 through the activation of CD4-positive T lymphocytes.