An in vitro infection system of Trypanosoma cruzi and HeLa cells was used to measure the anti-T. cruzi activities of various calcium antagonists classified into dihydropyridines, diphenylalkylamines, and benzothiazepines and of allopurinol and benznidazole as medium and highly effective reference compounds, respectively. Six dihydropyridines (10 μM each), i. e. nifedipine, nicardipine, nimodipine, nisoldipine, nitrendipine, and amlodipine, decreased the rates of infection of HeLa cells from 11.7% (control) to 5.8, 0.9, 1.2, 3.6, 5.9, and 1.7%, respectively. Nicardipine and amlodipine were highly toxic to HeLa cells, causing detachment of cells from coverslips. Nimodipine was thus the most effective inhibitor tested against T. cruzi infection in HeLa cells. Verapamil and gallopamil (diphenylalkylamines), diltiazem and midazolam (benzothiazepines), and allopurinol (positive control) were less effective than nimodipine. IC₅₀ values, the concentrations of compounds that elicited a 50% reduction in the infection rates of HeLa cells, were 2.5, 2.6, 1.3, 2.1, and 1.7 μM for nicardipine, nimodipine, amlodipine, verapamil, and benznidazole, respectively, while the values for nifedipine, diltiazem, and allopurinol were much higher. Nicardipine, amlodipine, and verapamil again showed significant cytotoxicities to HeLa cells. When Swiss 3T3 fibroblasts replaced HeLa cells, nimodipine markedly lowered the host-cell-infection rate, with an IC₅₀ value of 8.3 nM. Thus, nimodipine is expected to be a highly effective anti-T. cruzi lead compound, with low cytotoxicity to mammalian cells. Structural formulas of nimodipine and nicardipine in relation to their low and high cytotoxicities, respectively, against HeLa cells are discussed.