Cultured rat embryo fibroblasts, when placed in media with 10% serum containing 20 mM NH4Cl, show an inhibition of protein degradation and, concurrently, an accumulation of numerous, large vacuoles, partially filled with cellular debris. Cells placed in a serum-free media exhibit an enhanced degradation of cell protein, which is also inhibited by NH4Cl. When these cells are removed from media containing NH4Cl and placed in fresh media, the material accumulated in these vacuoles is rapidly and quantitatively released to the media in both an acid-soluble and acid-insoluble from. NH4Cl inhibits rapidly and specifically the lysosomal proteolytic mechanism, and is without effect on the basal turnover mechanism. The lysosomal proteolytic mechanism accounts for approximately 25% of protein turnover, and, at least in low density cultures, can be stimulated to levels which account for more than half of the protein turnover in the cell. The major pathway for the degradation of fast turnover proteins appears to be separate from lysosomal mechanism.