The cellular factors that regulate infection and replication of respiratory syncytial virus (RSV) in human alveolar macrophages were examined. RSV-exposed alveolar macrophages demonstrated a time-dependent expression of viral glycoproteins, maximal by 24 h post-infection resulting in infection of approx. 38% of the cells. Essentially all (33%) of these freshly isolated alveolar macrophages replicated RSV as shown by infectious centre assays. This RSV-permissive subpopulation of alveolar macrophages consisted primarily of major histocompatibility class II-expressing cells as determined by fluorescence-activated cell sorting. Re-infection of alveolar macrophages did not significantly alter the number of cells infected or capable of replicating RSV. However, in vitro differentiation of alveolar macrophages prior to infection resulted in a significant (P < 0.05), time-dependent decrease (approx. sevenfold) in the number of cells that replicated virus. The mechanism by which cellular differentiation restricted RSV replication is unknown. Production of defective interfering particles did not account for this decrease. Alveolar macrophages infected with RSV produce a variety of cytokines potentially contributing to this restricted viral replication. Pretreatment with several of these cytokines did not affect viral infection or replication. However, tumour necrosis factor (TNF alpha) significantly (P < 0.05) decreased viral replication but only by 30 to 60%. Thus RSV replication is reduced by in vitro differentiation of alveolar macrophages and, to a lesser degree, by pretreatment with TNF.