The intracellular survival of the ubiquitous pathogen Listeria monocytogenes was studied in primary cultures of bone marrow-derived mouse macrophages. Bacteria were able to grow rapidly in these cells, with an apparent multiplication rate of about 40 min. Electron microscopy demonstrated that intracellular bacterial replication was the consequence of simultaneous intracellular killing and replication of bacteria in the same cells. Within the first hour following phagocytosis, most bacteria were destroyed in the phagosomal compartment to which they were confined. This was due to early transfer of hydrolytic enzymes to phagosomes, undoubtedly via phagosome-lysosome (P-L) fusion, as demonstrated by a quantitative analysis after staining for a lysosomal marker, acid phosphatase. One hour after infection, about 14% of the bacteria were free in the cytoplasm, in which they multiplied and induced actin polymerization and spreading to adjacent macrophages, as in epithelial cells. By using the 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine staining procedure, direct evidence is presented that all phagosomes were acidified immediately after phagocytosis, thus indicating that intraphagosomal bacteria were exposed to an acidic environment that might favor vacuolar lysis by listeriolysin O. Intracellular growth in macrophages, therefore, appears to be the result of a competition between the expression of the hydrolytic activity of these cells following P-L fusion and the capacity of L. monocytogenes to escape from the acidified phagosomal compartment before P-L fusion has occurred. The finding that concomitant intracellular killing and survival of L. monocytogenes occurs in the same macrophages might explain the high immunogenicity observed in vivo with live bacteria, as opposed to killed bacteria.