During virus assembly, a subset of human immunodeficiency virus (HIV) matrix (MA) molecules is phosphorylated on C-terminal tyrosine. This modification facilitates infection of nondividing cells by allowing for the recruitment of the karyophilic MA into the viral core and preintegration complex. MA tyrosine phosphorylation is accomplished by a cellular protein kinase which is incorporated into virions. In this study, we have investigated the nature of this enzyme as well as the determinants of MA necessary for its phosphorylation. Employing an in vitro kinase assay, we found that the MA tyrosine kinase activity is present in various cultured cell lines including CEM and SupT1 T-lymphoid cells, Namalwa B cells, 293 and CV-1 kidney fibroblasts, and P4 HeLa cells. In addition, it could be detected in platelets, macrophages, and activated peripheral blood lymphocytes (PBLs) but not in erythrocytes and resting PBLs isolated from human blood. Subcellular localization of the kinase activity by cell fractionation demonstrated that it is enriched in cellular membranes. In HIV type 2 (HIV-2) particles, the MA tyrosine kinase is associated with the inner leaflet of the viral membrane, while the tyrosine-phosphorylated MA is localized to the core. Individual mutations of each of the last eight residues immediately upstream of the C-terminal tyrosine (Y132) of HIV-1 MA did not prevent Y132 phosphorylation, suggesting that the kinase does not require a highly specific sequence adjacent to the C-terminal tyrosine. Confirming this, we found that the MA of murine leukemia virus, the sequence of which is only moderately homologous to that of HIV-1 and HIV-2 MA, is also C-terminally tyrosine phosphorylated.