A method for the analysis of androsterone sulfate in serum by means of an enzymatic cycling reaction was developed. Androsterone sulfate was solvolyzed, extracted and purified on Sephadex LH-20, then androsterone was oxidized by 3α-hydroxysteroid dehydrogenase from Pseudomonas tetstosteroni and the resulting NADH was determined by an enzymatic cycling method. The cycling system consisted of two enzyme, alcohol dehydrogenase from yeast and diaphorase from Clostridium kluyverii, and resazurin was used as an electron acceptor for diaphorase. Subsequently, resorfin formed from the resazurin was determined by the rate assay method at Ex₅₆₀, Em₅₈₀ and 25°C. A linear relationship was obtained between the amount of androsterone and ΔF/min in the range from 0.035 to 3.5 μg/ml. The recovery, the reproducibility and the correlation between the proposed method and the conventional method using gas-liquid chromatography were also good. It appears that this simple and sensitive method could be useful as a clinical diagnostic test.