Deletion mutations at the C terminus of the matrix (MA) protein of human immunodeficiency virus type 1 (HIV-1) were generated by site-directed mutagenesis. The resultant mutant viruses had a severe defect in virus infectivity. This defect did not involve late steps of the virus life cycle, as the synthesis and processing of the Gag polyprotein and the assembly and release of mutant virions were not greatly affected. The incorporation of viral proteins and the viral RNA genome was similar for mutant and wild-type virions. In contrast, the early steps of the virus life cycle were severely affected, as the synthesis of viral DNA postinfection was dramatically reduced in mutant-virus-infected cells. One stretch of amino acids that was deleted in one of the mutants has significant homology with a region in VP1 of the picornavirus family. This region of VP1 is presumably involved in poliovirus penetration into cells. These results suggest that in addition to its functional role in virus assembly, the MA protein of HIV-1, and possibly of other retroviruses, plays an important role in virus entry.