B16 murine melanoma melanosomes were purified using sucrose density gradient centrifugation. ATPase activity was evaluated in presence of specific ATPase inhibitors, and compared with melanosome ATP-driven proton translocating activity in the melanosome. Mg2+ dependent ATPase activity was greatly inhibited (82%) by the specific inhibitors of vaculor proton translocating ATPase; Cis-didimethylsulfoxide dichloroplatinum (II) at approximately 90 microM and bafilomycin AI at two fold higher concentrations. Less inhibition, about 30 and 45% was obtained with N, N1-dicyclohexylcarbodiimide and N-ethylmaleimide, and the maximal effect occurred in the 50-100 microM and 0.1-1.5 mM ranges, respectively. These drugs at similar concentrations also inhibited the proton pumping activity to the same extent as observed for ATPase activity and half-maximal inhibition of each activity was found at nearly similar concentrations. Carbonylcyanide p-trifluoromethoxyphenyl hydra zone (FCCP) prevented ATP from setting up a pH gradient across the melanosomal membrane but stimulated Mg2+ ATPase activity significantly. Replacement of 5 mM Mg2+ with equimolar Ca2+ brought about a 60% inhibition in divalent cation-dependent ATPase- activity, and an 85% inactivation of ATP-linked melanosomal H+ pump activity. In the presence of optimal concentrations of Ca2+ and Mg2+ ATPase activity was similar to that seen in a Mg2+ medium. In Ca2+ medium ATPase activity was inhibited by CDDP and stimulated by FCCP, however these effects were two to three fold less than those observed in Mg2+ medium. FCCP failed to stimulate ATPase activity in CDDP- supplemented medium, thus suggesting that the same ATPase activity fraction was sensitive to both CDDP and FCCP. Mg2+-ATPase activity, like the proton-pump was anion dependent. The lowest activity was recorded in F medium, and increased in the order of F < So4(2-) < CL- = Br-. These results show that the ATPase activity may be related to the melanosomal proton pump.