Preliminary studies demonstrated that Clostridium sporogenes synthesized isoleucine by a pathway not involving threonine or threonine dehydratase. Radiotracer experiments with cells grown in a defined carbohydrate-free medium showed that radioactivity from [U-14C]serine, [3-14C]pyruvate, [14C]NaHCO3 and [1-], [2-] and [3-14C]propionate was incorporated into isoleucine. Conversely, there was no detectable incorporation of 14C into isoleucine during growth with [U-14C]glutamate, [U-14C]threonine, [U-14C]valine, [U-14C]leucine or [U-14C]methionine. Crude extracts of the bacteria grown in a minimal medium contained levels of alpha-acetohydroxyacid synthase activities comparable to those in Escherichia coli K12 grown in minimal medium. Stepwise degradation of isoleucine obtained from C. sporogenes grown in the presence of specifically-labelled precursors indicated that C. sporogenes can make isoleucine via the reductive carboxylation of propionate to yield alpha-oxobutyrate, which is metabolized to isoleucine in the classical fashion. Isoleucine was also formed by C. sporogenes via the reductive carboxylation of alpha-methylbutyrate to alpha-oxo-beta-methylvalerate.