Human immunodeficiency virus type 1 (HIV-1) is restricted for replication in rhesus macaque cells and does not establish infection in this species. The block to productive infection of macaque peripheral blood mononuclear cells (PBMC) in culture was investigated. A chimeric virus SHIV containing HIV-1 tat, rev, and env and all other genes from a simian immunodeficiency virus clone pathogenic in macaques (i.e., SIVmec239) replicated efficiently in macaque PBMC. Thus, the attachment step, involving interaction of the HIV-1 env glycoprotein with the cell surface CD4 receptor, is not blocked. Analysis of uptake of HIV-1 particles in these cells revealed a small reduction in virion entry; however, viral DNA synthesis, measured by PCR amplification, was greatly reduced. Taken together, these results indicate that the block to HIV-1 (subtype B) replication in rhesus macaque cells involves release of the virion core into the cytoplasm and/or a step immediately prior to initiation of reverse transcription. Further studies are required to characterize this block through identification of species-specific cellular proteins that interact with HIV-1 proteins in the early phase of viral replication.