A significant influence of dietary biotin has been observed in enhancing the blood, liver and fecal levels of unconjugated reduced folate cofactors in folate deficient rats. Total folate levels were, however, not influenced by dietary biotin. The observed decrease of liver folylconjugate synthetase activity in the presence of dietary biotin may plausibly explain the increased tissue levels of unconjugated folates in biotin fed rats.