The present work reports the in vitro biological characterization of 17 Trypanosoma cruzi isolates from southern and central México, and compares these results to those of four South American strains and one clone from Brazil. The parameters evaluated were growth rates, percentage of parasites undergoing transformation from epimastigotes to trypomastigotes, infectivity to, and in vitro killing of cultured Vero and P388 cells. Isoenzyme patterns of 11 enzymatic systems and 16 loci were also determined for the Mexican isolates. The parasites showed differences in growth, depending if they were cultured in LIT with hemin or in Grace's media. Transformation was obtained only in Grace's medium and differences were observed between the stocks. Stocks Z10 and Z21 showed the highest percentage of transformation within the Mexican isolates (39 and 41%, respectively). A second group showed percentages of transformation between 15 and 28%. In contrast, the South American strains showed higher rates of transformation (36-65%). Infection of cultured cells by isolates Z10 and H5 was evaluated in both Vero and P388 cells. Differences were observed both in the percentage of infected cells as well as in the number of amastigotes per cell. Differences in the ability to cause in vitro killing of P388 cells were also observed among the isolates. Isoenzyme analysis revealed genetic variation between the isolates, each of them with an unique zymodeme. This genetic analysis revealed, in general, a clustering based on the geographical origin of the isolates. Finally, correlation with clinical symptoms is discussed.