The proinflammatory cytokine tumor necrosis factor alpha (TNFalpha) is readily detected after human immunodeficiency virus type 1 (HIV-1) infection of primary macrophages in vitro and is present in plasma and tissues of patients with AIDS. Previous studies have shown that human recombinant TNFalpha (hrTNFalpha) enhances HIV replication in both chronically infected promonocytic and T-lymphoid cell lines in vitro. We report here that in contrast to untreated tissue culture-differentiated macrophages (TCDM), in which the proviral long terminal repeat (LTR) could be detected as soon as 8 h postinfection by a PCR assay, TCDM pretreatment for 3 days by hrTNFalpha markedly delayed its appearance until 72 h after infection with the HIV-1 Ada monocytotropic strain. Moreover the inhibition of formation of the proviral LTR in HIV-1-infected TCDM was directly proportional to the concentration of hrTNFalpha used. To determine if the inhibition of LTR formation results from blockade of viral entry, we performed a reverse transcription PCR assay to detect intracellular genomic viral RNA as early as 2 h after infection. Pretreatment of primary TCDM by hrTNFalpha for 3 days and even for only 2 h inhibits 75% of the viral entry into the cells. The inhibition of viral entry by hrTNFalpha was totally abolished by the use of anti-human TNFalpha monoclonal antibody. By using TNFalpha mutants specific for each human TNFalpha receptor, we showed that the inhibition of HIV-1 entry into TCDM was mediated not through the 55-kDa TNF receptor but through the 75-kDa TNF receptor. Although prolonged (1 to 5 days) TNFalpha treatment can downregulate CD4 expression in primary human TCDM, surface CD4 levels were not reduced by 2 h of treatment and was therefore not a limiting step for HIV-1 entry. In contrast to the inhibition of viral entry into primary TCDM, pretreatment with hrTNFalpha did not modify HIV-1 entry into phytohemagglutinin A-activated peripheral blood lymphocytes. TNFalpha-pretreatment inhibited HIV-1 replication in primary TCDM but not in phytohemagglutinin A-activated peripheral blood lymphocytes as assessed by decreased reverse transcriptase activity in culture supernatants. These results demonstrate that TNFalpha is able to enhance host cellular resistance to HIV-1 infection and that selective inhibition of HIV-1 entry into primary TCDM by TNFalpha involves the 75-kDa TNF receptor but not the 55-kDa TNF receptor.