Human sterol 27-hydroxylase (CYP27A1) has been studied in the present thesis with focus on substrate specificity and possible role of the enzyme for prevention of accumulation of cholesterol and cholestanol in tissues. Attempts have been made to evaluate a metabolic pathway to bile acids in human primary hepatocytes that starts with a 27-hydroxylation of cholesterol catalyzed by CYP27A1. The first Swedish case of sterol 27-hydroxylase deficiency (Cerebrotendinous xanthomatosis, CTX) is described and the mutation in the CYP27A1 gene is defined. Substrate specificity. Polar substrates were found to be 27-hydroxylated more efficiently than unpolar substrates by recombinant CYP27A1 or by cells overexpressing CYP27A1. A precursor to cholesterol, 7-dehydrocholesterol, was found to be a less efficient substrate for CYP27A1. Cholestanol, a 5a-saturated analogue to cholesterol, was found to be 27-hydroxylated by CYP27A1 at about the same rate as cholesterol. Cholestenone, a precursor to cholestanol, was found to be a highly efficient substrate for CYP27A1. The findings may be part of the explanation for the accumulation of cholestanol in patients with a sterol 27-hydroxylase deficiency (CTX). Cholestanol was also found to leave cultured cells at a slower rate than cholesterol, giving a partial explanation for its selective accumulation in CTX. Possible role in cholesterol accumulation. Cholesterolosis is a condition with accumulation of cholesterol esters in the mucosa of the gallbladder. CYP27A1 mRNA, CYP27A1 protein and CYP27A1 enzyme activity were found to be present in both normal gall bladder mucosa and in cholesterolosis in similar amounts. The esterifying enzyme ACAT was found to be upregulated in cholesterolosis, probably secondarily to the high cholesterol levels. The conclusion of the study is that CYP27A1 does not seem to be a pathogenic factor in cholesterolosis. Levels of esterified 27OH-cholesterol, the product of CYP27A1, were markedly increased in cholesterolosis, probably as a secondary effect of the ...