The possible role of intracellular Ca2+ level on Trypanosoma cruzi differentiation was explored. The addition to epimastigotes of a Triatoma infestans intestinal homogenate, which that triggers off the differentiation to the infective metacyclic form, induced a sudden rise in [Ca2+]i from the basal value, 94 +/- 28 to 584 +/- 43 nmole/liter. This increase was not affected by the presence of EGTA in the medium. Trypsin-treated intestinal homogenate did not alter the [Ca2+]i of epimastigotes. Calmodulin inhibitors (Calmidazolium, Trifluoperazine, and Chlorpromazine) blocked differentiation. Although the calcium ionophore ionomycin increased [Ca2+]i to 342 +/- 29 nmole/liter, it was unable to induce differentiation by itself. BAY K8644 and Methoxyverapamil (agonist and antagonist of Ca2+ channels, respectively) were unable to affect [Ca2+]i by themselves, or when added to stimulated parasites, and did not exert a stimulatory or inhibitory effect on morphogenesis. BAPTA/AM, a Ca2+ chelator, partially blocked the rise in [Ca2+]i and morphogenesis; this effect was reversed by ionomycin. The requirement of intracellular Ca2+ on epimastigote multiplication was also evaluated. The addition of EGTA to the culture medium led to a decrease in epimastigote multiplication till it practically ceased in the sixth passage. When such parasites were transferred to LIT they partially recovered the growth rate. Parasites from passages III, IV, and V in the Ca(2+)-depleted medium maintained their basal [Ca2+]i, but when treated with the intestinal homogenate, the rise in [Ca2+]i was abrogated. Accordingly, the differentiation percentages of such parasites dropped significantly compared with controls.