Production of methylthioribose by Escherichia coli was investigated under various growth conditions using sulfate as the exogenous source of sulfur. Methylthioribose was shown by tracer and chromatographic methods to be a normal biosynthetic product secreted into the medium and accumulated there. Production of [35S]-methylthioribose decreased by dilution of the radioactivity as total inorganic sulfate increased in a dense suspension of cells preincubated in sulfate-limiting medium. Variations in initial inoculum size had little effect on yield of this product.Formation of methylthioribose increased with decreasing levels of nitrogen under a sulfur deficiency, with decreasing levels of sulfur under a nitrogen deficiency, and with decreasing levels of phosphorus under a combined deficiency of sulfur and nitrogen. Methylthioribose increased in the alcohol-soluble, sulfur-containing fraction on prolonged incubation in phosphate-deficient, sulfate-deficient, and phosphate–sulfate deficient media. In the last two systems methylthioribose made up nearly 100% of the total fraction.In a survey of microorganisms using sulfate as the major source of sulfur, Escherichia coli produced methylthioribose to the greatest extent. Aerobacter aerogenes, Clostridium pasteurianum, and Saccharomyces cerevisiae produced only 2–3% as much. Bacillus subtilis, Staphylococcus aureus, Lactobacillus acidophilus, and Pseudomonas aeruginosa did not form detectable amounts of methylthioribose under conditions of this study.