5-S-Ethyl-5-thio-D-ribose (ethylthioribose) exhibits antiprotozoal activity against Plasmodium falciparum, Giardia lamblia, and Ochromonas malhamensis, but is nontoxic to cultured human and murine bone marrow cells (Riscoe, M. K., Ferro, A. J., and Fitchen, J. H. (1988) Antimicrob. Agents Chemother. 32, 1904-1906). We propose the following mechanism to account for the observed selective toxicity of ethylthioribose. 1) The cytocidal action of ethylthioribose against protozoa is a result of its conversion to ethionine, a well-known cytotoxic agent. 2) This transformation occurs through the pathway which normally converts 5-S-methyl-5-thio-D-ribose (methylthioribose) to methionine. 3) Conversion of ethylthioribose to ethionine cannot occur in mammalian cells since these cells cannot phosphorylate methylthioribose (ethylthioribose), a first step in the pathway to methionine (ethionine). To test this hypothesis, [5-3H]ethylthioribose has been synthesized and its metabolism by cell-free extracts of Klebsiella pneumoniae and rat liver was examined. The pathway by which methylthioribose is converted to methionine in K. pneumoniae is well characterized. When supplemented with ATP and L-glutamine, the bacterial extract efficiently converted [5-3H]ethylthioribose to [3H]ethionine. By contrast, ethionine was not produced upon incubation of [5-3H]ethylthioribose, ATP, and L-glutamine with rat liver homogenate. The mammalian cell extract lacks a kinase activity capable of converting ethylthioribose to 1-phospho-5-S-ethyl-5-thio-alpha-D-ribofuranoside, an obligate intermediate in the biosynthesis of ethionine from ethylthioribose in K. pneumoniae. These results support our hypothesis and provide a basis for understanding the apparently selective toxicity of ethylthioribose.