The gag proteins of HIV-1 are modified by the addition of myristic acid to the amino terminal glycine residue. Site-directed mutagenesis was used to construct a mutant of HIV-1 in which this glycine residue was changed to an alanine. Upon transfection into cos-1 cells, the mutant genome directed the synthesis of the full complement of HIV-1 proteins, but p17 and p17-containing polyproteins were not myristoylated. The cells transfected with the mutant DNA did not release any virus particles and no viral cores were visible by electron microscopy. Furthermore, supernatant from these transfected cells failed to infect CEM cells. The expression and function of gp120 on the surface of cells transfected with the mutant DNA was unaffected as these cells formed syncytia comparable in both size and number to the ones obtained with wild-type DNA.