A simple and sensitive method was developed for the quantification of serum total 3alpha-hydroxy bile acids. 0.1 ml of serum was mixed with tris(hydroxymethyl) aminomethane hydrochloric acid buffer and heated at 67 degrees C for 30 min. To the solution were added 3alpha-hydroxysteroid : oxidoreductase (EC 18.104.22.168; 3alpha-HSD), NAD, diaphorase (EC 22.214.171.124) and resazurin. The mixture was incubated at 20 degrees C for 1 h. The resultant fluorescence of resorfin was measured at 580 nm with the excitation at 560 nm. The blank value was obtained after the same treatment of another 0.1 ml of the same serum without 3alpha-HSD. A linear relationship was obtained between the amount of bile acids and the fluorescence intensities in the range of 1 to 150 mumol/1. The recovery of bile acids added to the serum was 81.4 +/- 2.5 (S.D.)% for cholate, chenodeoxycholate and deoxycholate. The bile acid content in the serum was 48.8 mumol/1 with a standard deviation of +/- 0.42 and a coefficient of variation of +/- 0.87% in 10 replicate determinations. The mean bile acid content of normal fasting male sera was 8.0 mumol/1 (3.6-12.6 mumol/1, n = 12) and of female sera 6.8 mumol/1 (3.2-12.7 mumol/1, n = 13).