To test whether in vitro infection of macrophages with either human immunodeficiency virus (HIV) or mycobacteria would influence the replication of the other pathogen, macrophages were infected sequentially with the macrophage-tropic isolate HTLV-IIIBa-L/85 and Mycobacterium tuberculosis H37Rv or Mycobacterium avium. The intracellular growth of mycobacteria was measured by colony counting and radiometric assay of macrophage lysates and the replication of HIV by the release of p24 antigen into the culture supernatants. Phagocytosis and intracellular growth of mycobacteria was similar in HIV-infected macrophages and controls. Conversely, mycobacteria did not affect the replication of HIV in macrophages. These experiments failed to demonstrate any direct intracellular interaction between HIV and mycobacteria in cultured macrophages that would explain the increased rate of mycobacterial diseases in patients infected with HIV or that would support the hypothesis that mycobacterial infection of macrophages per se can enhance HIV replication in these cells.