When KB cells labeled with 51Cr (1 muCi/ml) were treated with adenovirus type 2 (Ad2) at pH 6, 51Cr was released in a concentration-dependent manner with half-maximal release at 1 microgram/ml of virus. The 51Cr release was maximum at 10 micrograms/ml and represented nearly 20% of cell-associated 51Cr. 51Cr release depended on the length of incubation with Ad2 and the pH of the medium; maximum release was at pH 6 with very little release at pH 7.5. An antiserum against the penton base of Ad2 specifically neutralized the ability of Ad2 to release 51Cr. Chloroquine up to 200 microM did not block Ad2-dependent 51Cr release. DEAE-dextran stimulated Ad2-dependent 51Cr release; 4-5-fold stimulation was observed at 50 micrograms/ml of DEAE-dextran. We have compared the ability of Ad2 to release 51Cr with its ability to disrupt endocytic vesicles. Vesicle disruption was independent of pH of the medium in the range of pH 6 to 7.5 and was blocked by chloroquine, whereas, 51Cr release was much greater at pH 6 than at pH 7.5 and was not affected by chloroquine. These results suggest that Ad2 possess a lytic activity which ordinarily lyses acidic endocytic vesicles but, if the cells are maintained at acidic pH, can also directly disrupt the plasma membrane.