ATP-driven acidification of internal compartments of Trypanosoma cruzi epimastigotes was assayed spectrophotometrically with Acridine Orange and cells permeabilized with filipin. H+-ATPase activity was not inhibited fully by either 500 nM concanamycin A or 500 microM orthovanadate, but a combination of 5 nM concanamycin A and 25 microM vanadate completely inhibited activity, suggesting the operation of separate V-type (concanamycin-sensitive) and P-type (vanadate-sensitive) H+-ATPase activities in the permeabilized cells. This was supported by different kinetics of Acridine Orange uptake seen in the presence of the different inhibitors, and by different optimal protein (cell) concentrations for the two apparent activities. The use of different buffers further distinguished the ATPases. The V-H+-ATPase activity was stimulated by K+ and inhibited by a lack of anions or the replacement of Cl- with gluconate. The P-type H+-ATPase activity was not affected by a lack of Cl- or K+ but was substantially inhibited in a largely anion-free buffer. This inhibition could be annulled by the addition of the K+ ionophore valinomycin, which probably acted via the establishment of a countercurrent efflux of K+ from the compartment containing the P-type H+-ATPase and the relief of the potential difference generated by the electrogenic proton pump. Valinomycin showed some stimulation of P-type activity in all buffers tested, but its effects on V-H+-ATPase activity were at best transient except in a K+-free buffer, which suggested that the V-H+-ATPase was located in an organelle with relatively low [K+] that was different from that which accommodated the P-type activity. On the basis of acidity and K+ content, these organelles might correspond, in part at least, to the acidocalcisomes (V-H+-ATPase activity) and the reservosomes (P-type activity) previously identified in these cells. Both activities could also be found in the human-infective forms of the parasite, amastigotes and trypomastigotes, but the P-type activity was relatively weak in these cells types, which is correlated with a lack of reservosomes in these forms.