Anticoccidial drugs were evaluated for activity and for the development of resistance in a model of Toxoplasma gondii growing in human fibroblast cultures. Of 13 anticoccidial drugs tested, 9 had selective antitoxoplasma activity (50% inhibitory concentration, in micrograms per milliliter): decoquinate (0.005), arprinocid-N-oxide (0.015), robenidine (0.03), the aryl triazine CP-25,415 (0.2), toltrazuril (0.4), clopidol (1), dinitolmide (Zoalene; Dow) (10), and the carboxylic acid ionophores monensin (0.001) and salinomycin (0.04). Glycarbylamide, amprolium, nicarbazin, and the 6-(p-bromophenoxy)-7-chloro analog of halofuginone (Stenorol; Roussel-UCLAF) (CP-63,567) were toxic for the fibroblasts. Since Eimeria tenella has a similar drug susceptibility profile, anticoccidial drugs can be viewed as a potential source of new antitoxoplasma therapies. The development of resistance has limited the usefulness of most of these drugs as anticoccidial agents; in coccidia, resistance to all except the ionophores occurs readily in vivo. We explored the development of resistance in T. gondii by attempting to select mutants in vitro from parasites mutagenized with ethylnitrosourea. Mutants that had 20- to 50-fold-reduced susceptibility to decoquinate, arprinocid-N-oxide, and CP-25,415 were obtained. Ionophore-resistant T. gondii mutants were also selected in vitro; however, there was only a twofold difference in susceptibility between these mutants and the wild type. For three drugs (clopidol, robenidine, and toltrazuril), we were unable to select resistant mutants. For experimental anticoccidial drugs, there is currently no in vitro method for assessing the risk of development of resistance in Eimeria species. Our results suggest that T. gondii may offer a useful surrogate for this assessment.