Recent development in research concerning hydroxylations in biosynthesis and metabolism of bile acids is reviewed. In the conversion of cholesterol into bile acids, hydroxyl groups may be introduced into the following positions: C-6α, C-6β, C-7α, C-12α, C-16α, C-23, C-24, C-25, and C-26. With the exception of the 26-hydroxylation and probably the 24-hydroxylation, which are also catalyzed by the mitochondrial fraction, the different hydroxylations are catalyzed by the microsomal fraction of liver homogenate and require NADPH and molecular oxygen. With the possible exception of the 12α-hydroxylase, all the microsomal hydroxylations appear to involve participation of cytochrome P-450 and NADPH-cytochrome P-450 reductase. Several of the hydroxylations have been demonstrated in reconstituted systems consisting of partially purified cytochrome P-450, NADPH-cytochrome P-450 reductase and a phospholipid. The specificity of the hydroxylation is determined mainly by the cytochrome P-450 fraction. The cholesterol 7α-hydroxylase catalyzes the major rate-limiting step in the overall conversion of cholesterol into bile acids. The combined activities of the microsomal 26-hydroxylase and the 12α-hydroxylase play a major role in determining the ratio between cholic acid and chenodeoxycholic acid formed from cholesterol. There is a close relationship between the cholesterol 7α-hydroxylase activity and the rate of cholesterol biosynthesis. The regulatory role of the cholesterol 7α-hydroxylase, the 12α-hydroxylase and the microsomal 26-hydroxylase may be correlated with some specific properties of the enzyme systems which differ markedly from the properties of the other hydroxylases involved in biosynthesis and metabolism of bile acids. The influence of different factors on the hydroxylations has been studied and the physiological implications of the different effects are discussed.