Within minutes after the onset of deprivation of an essential nutrient, all bacteria develop resistance to lysis by beta-lactam antibiotics, a phenomenon termed phenotypic tolerance. Two phases of this process were identified in pneumococci and the activity of the major autolysin, an N-acetylmuramyl-L-alanine amidase, was studied in each phase. Autolysin was detectable by immunofluorescence in a uniform distribution over the surface of growing pneumococci, but became progressively depleted during amino acid deprivation. Lysis of nongrowing cells by beta-lactam antibiotics could be reconstituted by addition of exogenous autolysin during the first 80 minutes of starvation (Phase I) but not thereafter (Phase II). Similarly, Triton X-100 or deoxycholate lysed nongrowing cells in Phase I but not Phase II. Cell wall isolated from Phase II cells was found to be more resistant to hydrolysis by the autolysin in vitro than that from growing cells. Lysis of growing cells could also be inhibited by incorporation of a pulse of nonhydrolysable cell wall or autolysin deficient cell wall into the growth zone. These results suggest that phenotypic tolerance in nongrowing pneumococci involves rapid loss or disengagement of autolysin molecules from their in situ attack-sites (Phase I) followed by a second slower process that involves a progressive change in the cell wall structure to a form less susceptible to hydrolysis by the autolysin (Phase II).