Murine fibroblasts expressing viral receptors and human cyclin T1 allow HIV-1 entry and viral gene expression but do not support efficient assembly. A chimeric HIV-1 carrying a non-homologous matrix (MA) from murine leukemia virus in place of HIV-1 MA can assemble efficiently in murine cells, yet has poor infectivity. Here, we assess the ability of a homologous MA from SIV MAC239 to complement assembly and infection in chimeric viruses designated SHIV(MA). The resulting SHIV(MA) chimeras produce more virus than native HIV-1 when transfected into murine cells. SHIV(MA) exhibits cell-type-specific replication in human T cell lines, replicating well in MT4 cells and poorly in Jurkat cells due to an incompatibility with the HIV-1 Env. The infectivity defects of SHIV(MA) are rescued by pseudotyping with VSV-G but not by truncation of the cytoplasmic tail of Env. Passage of SHIV(MA) in Jurkat cells produces variants with improved Env incorporation and improved replication in Jurkat but not in 3T3 TXC cells. The results indicate that cell-type-specific, or species-specific, host factors interact with MA to modulate the efficiency of assembly and its compatibility with Env. With additional selection, SIV/HIV-1 chimeras may be useful for the development of murine models of lentiviral infection.