The LD50 of strains of L. interrogans used in the potency assay of the Cuban trivalent leptospiral vaccine intended for human use was standardized. The control of the leptospires content of the cultures by means of the adjusting dilution (10 to 12 leptospires per field) was introduced. That was the key of the standardization of the assay, since it guaranteed the reproducibility of the estimate of the LD50 value for each serogroup: 10(5.98) for canicola, 10(5.60) for icterohaemorrhagiae and 10(6.60) for pomona, with the following limits for an interval of 1 log10: 10(4.98)-10(6.98), 10(4.60)-10(6.60) and 10(5.49)-10(7.49), respectively; whereas for a 95% (+/- 2 SD) confidence interval the values were: 10(5.54)-10(6.19); 10(5.33)-10(5.75) and 10(6.35)-10(6.60), respectively. These values were lower than the previous ones, which means ever more reproducibility. The methodology described proved to be satisfactory for the standardization of the LD50 of the challenge strains and it could also be used for evaluating the virulence of other strains of L. interrogans, including those used for the vaccine production.