Isozyme patterns of 13 enzymes were compared for cultures of Trypanosoma avium, T. vespertilionis, T. cruzi and T. rangeli. The isozyme separation was made by cellulose acetate electrophoresis. Each of the species had distinctly migrating isozyme bands for glutamate oxaloacetate transaminase (GOT), isocitrate dehydrogenase (ICD), malic enzyme (ME), 6-phosphogluconate dehydrogenase (6PGD), phosphoglucoisomerase (PGI), phosphoglucomutase (PGM), and malic dehydrogenase (MDH). For other enzymes, two or more species had identically migrating bands. In addition to these interspecific species differences, variability was observed among the strains of T. cruzi and T. rangeli. Among the T. cruzi strains, there were two different isozyme (possibly allozyme) types of the enzymes alanine aminotransferase (ALAT), fructokinase (FK), glucose-6-phosphate dehydrogenase (G6PDH), GOT, MDH and three types of ME. In the T. rangeli isolates two isozyme types for the enzymes ALAT, FK, G6PDH, GOT, ICD, and LDH, were observed. Among the eight strains of T. cruzi studied there were six isozyme types, and among the seven T. rangeli isolates there were four isozyme types. There was an indication that isozyme types were associated with geographical distribution.