In 1891 Guttmann and Ehrlich (P. Guttmann and P. Ehrlich, Berlin Klin. Wochenschr. 28:953-956, 1891) were the first to report the antimalarial properties of a synthetic, rather than a natural, material when they described the clinical cure of two patients after oral administration of a thiazine dye, methylene blue. Since that time, sporadic reports of the antimalarial properties of several xanthene and azine dyes related to methylene blue have been noted. We report here the results from a reexamination of the antimalarial properties of methylene blue. Janus green B, and three rhodamine dyes and disclose new antimalarial data for 16 commercially available structural analogs of these dyes. The 50% inhibitory concentrations for the chloroquine-susceptible D6 clone and SN isolate and the chloroquine-resistant W2 clone of Plasmodium falciparum were determined by the recently described parasite lactate dehydrogenase enzyme assay. No cross-resistance to chloroquine was observed for any of the dyes. For the 21 dyes tested, no correlation was observed between antimalarial activity and cytotoxicity against KB cells. No correlation between log P (where P is the octanol/water partition coefficient) or relative catalyst efficiency for glucose oxidation and antimalarial activity or cytotoxicity was observed for the dyes as a whole or for the thiazine dyes. The thiazine dyes were the most uniformly potent structural class tested, and among the dyes in this class, methylene blue was notable for both its high antimalarial potency and selectivity.