During the process of phagocytosis, polymorphonuclear leukocytes (PMN) release lysosomal enzymes into the extracellular medium. When the antibiotic cytochalasin B (CB) is present in the incubation medium along with phagocytable particles, enhanced recovery of enzyme activities from the incubation medium has been observed. These findings have led to the interpretation that CB enhances lysosomal enzyme release. Our results contradict this interpretation. The lysosomal enzymes acid phosphatase and beta-galactosidase are unstable after they are released from cells. During the first 5-15 min of phagocytosis, significant amounts of both acid phosphatase and beta-galactosidase can be recovered from the extracellular medium. After this, the recovery of enzyme from the medium declines, presumably because the rate of loss of lysosomal enzyme activity exceeds the rate of release at later time periods. In the presence of CB, the appearance of lysosomal enzymes in the extracellular medium of cells exposed to zymosan is retarded for 5-10 min, after which it begins and then continues for approximately 20 min. At the end of a 30-min incubation period, therefore, in the absence of CB, extracellular levels of lysosomal enzymes (especially those which are unstable) are declining toward low levels while, in the presence of CB, extracellular enzyme levels are continuing to rise. We also measured the lysosomal enzyme remaining within cells after exposure to zymosan. CB retarded the disappearance of enzyme from cells and resulted in significantly less total cell enzyme loss. Thus, in the presence of CB, a greater proportion of the lysosomal enzyme lost from cells is recovered in the extracellular medium. In contrast to the previous conclusions that CB enhances lysosomal enzyme release, our results indicate that CB delays and decreases the zymosan-stimulated release of lysosomal enzymes from PMN. Since CB inhibits phagocytosis by PMN, our results indicate that the antibiotic modifies the mechanism of release of lysosomal enzymes, resulting in zymosan stimulation of their release independently of phagocytosis.