A mutant strain of Escherichia coli K-12 requiring lipoic acid. W1485lip 2 (ATCC 25645), was used to develop a turbidimetric assay for lipoic acid and a polarographic assay based on the oxidation of pyruvate by suspensions of lipoic acid-deficient organisms. The turbidmetric assay was more sensitive with a working range equivalent to 0.2-2.0 ng of DL-alpha-lipoic acid compared with 5-50 ng for the polarographic method. The mutant responded equally to racemic mixtures of alpha-lipoic acid, beta-lipoic acid and dihydrolipoic acid but gave little response to lipoamide, and other derivatives without prior hydrolysis; 8-methyllipoic acid was a competitive inhibitor of the response to lipoic acid. A high specificity of the mutant for the natural steroisomer was indicated by the fact that (+)-alpha-lipoic acid had twice the activity of the racemic mixture. Escherichia coli K12 contained less than 0.05 ng of free (+)-alpha-lipoic acid per mg dry weight but, depending on the growth substrate, the equivalent of between 13 and 47 ng of (+)-alpha-lipoic acid per mg dry weight after acid extraction. There was a strong correlation between the lipoic acid content and the sum of the specific activities for the pyruvate and alpha-ketoglutarate dehydrogenase complexes. Experiments with washed suspensions of Escherichia coli showed only small increases in lipoic acid content (18%) when incubated with pyruvate, cysteine and methionine. When supplied with exogenous lipoic acid the mutant, W1485lip2, accumulated very little more than was demanded by its metabolism. The lipoic acid contents of several organisms were measured and correlated with their metabolism.