The stereochemical course of reduction of crotonyl CoA by the novel crotonyl CoA reductase (CCR) of Streptomyces collinus was determined using a radiochemical assay. The reaction was shown to proceed with transfer of the hydrogen from the pro-4S position of NADPH to the Re face of the beta-carbon of crotonyl CoA. This transfer represents the first exception to the observation that enoyl thioester reductases catalyze transfer of the pro-4S hydrogen of NADPH to the Si face of the substrate. The observation of addition of solvent hydrogen to the Re face of the alpha-carbon in the reaction catalyzed by CCR demonstrated that the overall reduction of crotonyl CoA proceeds in an anti fashion. The overall stereochemical outcome of the reaction catalyzed by CCR is different to the four stereochemical outcomes that have previously been observed for enoyl thioester reductases. It is significant that the predicted amino acid sequence of CCR has also been shown to be unrelated to other enoyl thioester reductases. Based on these observations it is proposed that the stereochemical course of an enoyl thioester reduction serves no mechanistic function but merely reflects the pedigree or ancestral lineage of the enzyme. Any two enoyl thioester reductases which exhibit different stereospecificities are, therefore, predicted to have different pedigrees and unrelated amino acid sequences. An evaluation of all the enoyl thioester reductases where both the stereochemical course of reduction and the predicted amino acid sequence are known is shown to be entirely consistent with these predictions.