The major capsid (CA) protein of retroviruses possesses a stretch of 20 amino acids, called the major homology region (MHR), which is evolutionarily conserved and invariant in location within the primary sequence of the protein. The function of this region was investigated by examining the effect of random single-amino-acid substitutions within the central 13 positions of the MHR on the life cycle of Mason-Pfizer monkey virus (M-PMV), an immunosuppressive D-type retrovirus. When these mutants were subcloned into an M-PMV proviral vector and expressed in COS cells, one of two major phenotypes was observed. The first group, containing three mutants bearing drastic amino acid substitutions, was unable to assemble capsids in the cytoplasm of the host cell. The second and more common group of mutants was able to assemble and release virions, but these either displayed greatly reduced levels of infectivity or were completely noninfectious. Included within this second group were two mutants with unusual phenotypes; mutant D158Y exhibited a novel cleavage site for the viral protease that resulted in cleavage of the major capsid protein, p27 (CA), within the MHR, whereas mutant F156L appeared to have lost a major site for antibody recognition within the mature CA protein. The results of this mutagenic analysis suggest that changes in the MHR sequence can interfere with the assembly of viral capsids and block an early stage of the infection cycle of M-PMV.