The human parasite Toxoplasma gondii is sensitive to dinitroaniline compounds which selectively disrupt microtubules in diverse protozoa but which have no detectable effect on vertebrate host cell microtubules or other functions. Replication of wild-type T. gondii is inhibited by 0.5 to 2.5 microM oryzalin, but mutant parasites harboring amino acid substitutions in the predicted dinitroaniline binding site confer resistance up to 40 microM oryzalin. However, the precise interaction between dinitroanilines and the binding site in alpha-tubulin remains unclear. We have investigated the activity of 12 dinitroanilines and the related compound amiprophos methyl on wild-type and dinitroaniline-resistant parasite lines that contain proposed binding site mutations. These data indicate that dinitramine is the most effective dinitroaniline to inhibit Toxoplasma growth in wild-type parasites and most resistant lines. Dinitramine has an amine group at the meta position not present in any of the other dinitroanilines tested here that is predicted to form hydrogen bonds with residues Arg2 and Gln133 according to docking data. Remarkably, although the binding site mutation Ile235Val confers increased resistance to most dinitroanilines, it confers increased sensitivity to GB-II-5, a compound optimized for activity against kinetoplastid tubulin. Kinetoplastid parasites have a valine at position 235 of alpha-tubulin, whereas apicomplexan parasites have an isoleucine at this site. We suggest that this heterogeneity in binding site environment influences relative dinitroaniline sensitivity in distinct protozoan lineages and hypothesize that a mutation that makes the apicomplexan dinitroaniline binding site more like the kinetoplastid site increases sensitivity to a dinitroaniline optimized for activity in the latter parasites.