Using nonradioactive hybridization, the human multilocus probe 33.15 was shown to recognize multiple minisatellite regions in nuclear DNA from Trypanosoma cruzi, producing complex banding patterns on Southern blots, typical of DNA fingerprints. The DNA fingerprints were stable and were capable of identifying different strains of the parasite. Individual clones of the Y strain showed different banding patterns, demonstrating that the strain is heterogeneous. In general, the sensitivity and specificity of DNA fingerprinting was similar to that obtained with kinetoplast DNA restriction analysis. However, it has the advantages of being technically simple and of studying nuclear rather than mitochondrial DNA. Thus, it is a useful new tool for the characterization and study of strains and clones of Trypanosoma cruzi.