1. Thirty-eight steroids were tested as substrates for a 7 alpha-hydroxy steroid dehydrogenase preparation from a strain of Escherichia coli; an improved method of making the crude enzyme is described. 2. Steroids having a 7 alpha-hydroxyl group in the molecule were substrates except (a) when the 5 beta-cholan-24-oic acid side chain was shortened to less than four carbon atoms and (b) in certain cases when sulphate ester groups were present in the molecule. 3. For testing with the enzyme, a new specimen of 7 alpha-hydroxy-3,12-dioxo-5 beta-cholan-24-oic acid was made, which had properties different from those previously described.