The infectivity of Leishmania braziliensis ssp. in relation to their growth kinetics in Senekjie's medium was determined using the human macrophage cell line U937 and inbred hamsters. In both systems, infectivity was shown to be distinctive for each subspecies. While L. b. panamensis promastigotes from 6-day-old cultures (early stationary phase) were more infective than parasites from any other culture day, L. b. guyanensis and L. b. braziliensis reached maximum infectivity on days 8–10 and day 10 (late stationary phase of growth), respectively. Although maximum infectivity occurred during stationary growth, strict growth phase dependency was not observed. The populations of parasites on these culture days were composed mostly of small, highly motile promastigotes with flagella 2–3 times the length of their cell bodies. These promastigotes resembled the infective forms transmitted by the sand fly vector. A distinct pathological picture characterized the disease caused by the different WHO reference strains for these subspecies in hamsters: L. b. guyanensis developed the most severe lesions, while moderate and inconspicuous lesions were observed when L. b. panamensis and L. b. braziliensis, respectively, constituted the inocula.