The matrix domain of the Gag precursor protein, and the mature matrix protein, which is derived from processing of the Gag precursor, functions in several steps of the human immunodeficiency virus type-1 (HIV-1) life cycle. We made numerous mutations throughout the matrix protein and identified three mutants in the N-terminal portion of the matrix that drastically diminish the ability of the virus to replicate. Each of these replication-defective mutants was unable to acquire efficiently the envelope glycoprotein of HIV-1. To determine whether these same mutations affect other steps in viral replication we pseudotyped mutant particles with the envelope glycoprotein from an amphotropic murine leukemia virus. Each of these mutants was also hampered in other steps in virus replication. Two mutants were defective in entry or uncoating, and the third was hampered in a step following reverse transcription. Since viral replication was analyzed under conditions in which the nuclear localization function of the matrix protein is not required, the matrix protein may be required for an additional replication step following reverse transcription.