African sleeping sickness is a disease caused by Trypanosoma brucei. T. brucei proliferate rapidly in the mammalian bloodstream as long, slender forms, but at higher population densities they transform into nondividing, short, stumpy forms. This is thought to be a mechanism adopted by T. brucei to establish a stable host-parasite relationship and to allow a transition into the insect stage of its life cycle. Earlier studies have suggested a role for cAMP in mediating this transformation. In this study, using membrane-permeable nucleotide analogs, we show that it is not the cAMP analogs themselves but rather the hydrolyzed products of membrane-permeable cAMP analogs that prevent proliferation of T. brucei. The metabolic products are more potent than the cAMP analogs, and hydrolysis-resistant cAMP analogs are not antiproliferative. We further show that the antiproliferative effect of these membrane-permeable adenosine analogs is caused by transformation into forms resembling short, stumpy bloodstream forms. These data suggest that the slender-to-stumpy transformation of T. brucei may not be mediated directly by cAMP and also raise the possibility of using such adenosine analogs as antitrypanosomal drugs.