Human immunodeficiency virus type I (HIV- I) originated from three independent cross-species transmissions of simian immunodeficiency virus (SlVcpzPtt) infecting chimpanzees (Pan troglodytes troglodytes) in west central Africa, giving rise to pandemic (group M) and non-pandemic (groups N and 0) clades of HIV-1. To identify host-specific adaptations in HIV- I we compared the inferred ancestral sequences of HIV- I groups M, N and 0 to 12 full length genome sequences of SlVcpzPtt and four of the outlying but closely related SlVcpzPt.y (from P. t. schwehi irthii). This analysis revealed a single site that was completely conserved among SlVcpzPtt strains but different (due to the sarne change) in all three groups of HIV-1. This site, Gag-30, lies within p 17, the ga-encoded matrix protein. It is Met in SlVcpzPtt, underwent a conservative replacement by Leu in one lineage of SlVcpzPts but changed radically to Arg on all three lineages leading to HIV- 1. During subsequent diversification this site has been conserved as a basic residue (Arg or Lys) in most lineages of HIV- 1. Retrospective analysis revealed that Gag-30 had reverted to Met in a previous experiment in which HIV- I was passaged through chimpanzees. To examine whether this substitution conferred a species specific growth advantage, we used site-directed mutagenesis to generate variants of these chimpanzee-adapted HIV-1 strains with Lys at Gag-30, and tested their replication in both human and chimpanzee CD4+ T lymphocytes. Remarkably, viruses encoding Met replicated to higher titers than viruses encoding Lys in chimpanzee T cells, but the opposite was found in human T cells. Taken together, these observations provide compelling evidence for host-specific adaptation during the emergence of HIV- I and identify the viral matrix protein as a modulator of viral fitness following transmission to the new human host.