Post-mitochondrial supernatant extracts prepared from bloodstream forms of Trypanosoma brucei brucei, T. cruzi epimastigotes, Leishmania donovani promastigotes and Crithidia fasciculata have been found to catalyse cytochrome P450-dependent reactions. Appreciable ethoxycoumarin deethylase and ethoxyresorufin deethylase activities were found in all of the above trypanosomatids, with T. cruzi epimastigotes having the highest activity (57.1 and 10.7 pmol/min/mg protein, respectively). In all four species these reactions were inhibited by the cytochrome P450 inhibitors carbon monoxide, proadifen and metyrapone. In contrast to rat liver microsomes, the trypanosomatid extracts showed no detectable pentoxyresorufin depentylase or pentamidine hydroxylase activity. Both C. fasciculata and T. b. brucei post-mitochondrial supernatants showed carbon monoxide difference spectra consistent with the presence of cytochrome P450 (9.6 and 6.3 pmol/mg protein, respectively). An additional hemoprotein which gave a carbon monoxide difference peak at 420 nm was also detected in C. fasciculata and T. b. brucei microsomes and C. fasciculata mitochondria. Subcellular fractionation of both early and late log C. fasciculata showed that the ethoxycoumarin deethylase activity was enriched in the microsomal fraction.