The lysosomal/endosomal system of African trypanosomes is developmentally regulated and is important in the pathogenesis associated with infection of the mammalian bloodstream. Long considered to be a target for drug development, the internal pH of the lysosome has been variously reported to range from <5.0 to >6.0. We have refined a flow cytometric technique using a pH-sensitive probe that specifically targets the lysosome, tomato lectin:Oregon Green 488 conjugate. The probe is delivered to the lysosome with fidelity, where it is shielded against external pH. Measurement of fluorescent output in the presence and absence of lysomotropic agent (NH(4)Cl) then allows precise titration of steady state lysosomal pH (4.84+/-0.23). Using bafilomycin A1 to inhibit acidification we demonstrate that this method is responsive to pharmacological perturbation of lysosomal physiology. This work should facilitate future studies of the lysosomal function in African trypanosomiasis, as well as other parasitic protozoa.