The usual techniques for determination to total 3 alpha-hydroxy bile acids in serum involving liquid-solid extraction of the bile acids with the adsorbent XAD-2 and fluorimetric measurement of NADH generated from the reaction with a NAD-linked 3 alpha-hydroxysteroid dehydrogenase are evaluated and improved. The influence of different types of enzyme preparations on the results is examined. The results with the improved technique are compared to the results obtained with another method, avoiding extraction of the bile acids before the enzymatic reaction which is followed by fluorimetric measurement of resorufin, produced by transfer of the hydrogen of the generated NADH by diaphorase to resazurin. No significant difference between the results with the two types of methods was found. The concentration of total 3 alpha-hydroxy bile acids in serum of 46 fasting 'healthy' individuals aged 17 to 82 years is estimated. 30 were females, of whom 10 were taking estrogen-containing oral contraceptives, and 16 were males. Mean +/- standard deviation in all the females was 3.0 +/- 1.1 micromol/l, and in the males 4.0 +/- 1.9 micromol/l. There was no significant difference between any of the groups.