Serum ratios to cholesterol of lathosterol, and of cholestanol, campesterol and sitosterol measure respective relative cholesterol synthesis and absorption, but their clinical applicability is not known in evaluation of cholesterol metabolism under different dietary conditions. We compared relative synthesis and absorption of cholesterol to the respective absolute ones in healthy male volunteers (n 29) on four subsequent diets: baseline home (HD), low-cholesterol low-fat (LCLF), high-cholesterol low-fat (HCLF) and low-cholesterol high-fat (LCHF). Serum lipids, lipoproteins, sterols, fractional cholesterol absorption and sterol synthesis were examined. HCLF and LCHF decreased fractional cholesterol absorption by approximately 23–27 % from baseline HD (P < 0·05) and increased the levels of total and LDL-cholesterol in serum from LCLF by approximately 9–14 % (P < 0·05). On HCLF, bile acid synthesis was high (P < 0·05 for each), and absolute cholesterol synthesis tended to be higher than on HD and LCHF (NS). Relative synthesis was positively associated with absolute cholesterol synthesis, but inversely with relative absorption during each diet (P < 0·05). The relative absorption markers were interrelated in each diet, and were also associated with fractional absorption of cholesterol in each diet but HD. In conclusion, relative markers of cholesterol absorption and synthesis reflect changes in cholesterol metabolism despite the amount of dietary fat and cholesterol consumed, but their validity with this respect is strengthened by controlled diets in metabolic studies. Additions of cholesterol and fat to a diet low in fat and cholesterol cause practically equal changes in the serum lipid profiles, whereas synthesis of cholesterol (NS) and bile acids (P < 0·05) were higher with the high-cholesterol feeding.