1. Middle-repetitive DNA sequences were analyzed by molecular hybridization to determine both the extent of complementarity and time of evolutionary divergence between isolates of Trypanosoma cruzi from Argentina, Mexico, or Venezuela. 2. Although molecular hybridizations showed no significant difference between the middle-repetitive DNAs of the Mexican and Venezuelan isolates, there was a 2.7% base pair mismatch in hybrid molecules formed by association of strands from both the Mexican and Argentine isolates. 3. Using the rates for divergence of middle-repetitive DNA in sea urchins, the Mexican and Argentine isolates were estimated to have diverged approximately 20-25 million years ago. 4. Analysis of the kinetics for the DNA hybridizations indicates that only minor amplifications of specific gene sequences or changes in the complexity of the genomes could have occurred during the divergence of the three isolates studied.