The effect of biotin deficiency and supplementation upon tissue cholesterol and serum lipoprotein profile in rats was investigated. Biotin-dependent carboxylases in liver of deficient animals were decreased to 12-27% of control activity. The brain carboxylase activities of deficient rats were reduced less than in liver. The total, free, and esterified cholesterol were decreased in the serum of deficient rats compared to those in control or supplemented rats. Deficient rats had increased serum low-density lipoprotein fractions and decreased very low-density lipoprotein fractions, but no change in the high-density lipoprotein fractions. No differences were found between groups in the cholesterol content of liver, cerebrum, or cerebellum. Pharmacologic doses of biotin had no effect on tissue cholesterol content or serum lipoprotein profile. These results suggest that the neurologic symptoms in biotin deficiency and in the inherited multiple carboxylase deficiencies are not due to alterations in the content of cholesterol or lipoproteins.