HL60 cells isolated for resistance to vincristine (HL60/Vinc cells) or doxorubicin (HL60/Adr cells) contain enhanced levels of and energy-dependent drug efflux pump. HL60/Vinc cells contain the drug transporter P-glycoprotein, whereas the HL60/Adrisolate does not In the present study, we examined the possible involvement of vacuolar H + -adenosine triphos-phatase (H + -ATPase) activity in drug resistance in HL60 cells. We utilized bafilomycin A 19 , and agent which selectively inhibits vacuolar H + -ATPase activity at low concentrations. The results showed that bafilomycin A 1 induced a major increase in drug accumulation and inhibited drug efflux in both HL60/Adr cells and HL60/Vinc cells. Similar results were obtained with 7-chloro-4–nitrobenz–2–oxa 1, 3 diazole, and agent which is also capable of inhibiting vacuolar H + -ATPase. Azide, an inhibitor of F 1 F 0 mitochondrial ATPase, and vanadate and ouabain, which are inhibitors of E 1 E 2 -type ATPase, did not affect drug levels in resistant cells. We also observed that bafilomycin A 1 did not compete with ( 3 H)azidopine binding to P-glycoprotein. Thus, bafilomycin A 1 does not appear to function as a substrate for P-glycoprotein. These results suggest an involvement of vacuolar H + -ATPase activity in the pathway of drug efflux from HL60/Adr cells and HL60/brief-report Vinc cells. The mechanism of this action remains to be determined. [J Natl Cancer Inst 83: 1098–1102, 1991]