Cleavage of murine leukemia virus Pr65gag is associated with the activity of a labile proteolytic factor found in virions. We have shown that the presence of 80 to 100 mM NaCl inhibits this cleavage activity in vitro by over 90%. Further, the addition of 80 to 100 mM excess NaCl in vivo to chronically infected cultures of MJD-54 mouse fibroblasts also caused inhibition of Pr65gag cleavage. Specifically, the excess salt added to cells: (i) caused a greater than 90% decrease in virus production; (ii) increased the Pr65gag/p30 ratio in virions produced by more than threefold; and (iii) in pulse-chase experiments, showed a 10-fold decrease in the amount of Pr65gag cleaved after 3 h. In contrast, during this chase interval there was only a slight diminution, i.e., about two fold, in the cleavage of env precursor polyprotein Pr80env, suggesting that cleavages of Pr65gag and Pr80env are differently controlled. Additionally, electron microscopic examination of the excess salt-treated cells showed a twofold increase in the number of associated immature particles, consistent with the observed higher than average Pr65gag/p30 ratio. The inhibitory effects were also found if excess KCl or MgCl2 was used instead of NaCl, suggesting that they are caused by the hypertonic state of the medium and are not dependent on the ionic species used.