Because previous observations suggest that biotin may be present in vitamin preparations not labeled as containing biotin, we determined the biotin content of several over-the-counter vitamin and nutritional supplements said to contain biotin (group 1) and several in which biotin content was not specified (group 2). Biotin concentration was measured using the 125I-avidin assay which detects total avidin-binding substances. Water extracts were assayed for free biotin and acid hydrolysates were assayed for total biotin. The results of the 125I-avidin assay agreed with the stated biotin content for most vitamin and nutritional supplements in group 1. Biotin was the only avidin-binding substance in the preparations from group 1, based on reversed-phase chromatography. However, some vitamin and nutritional supplements in group 2 contained nutritionally significant amounts of biotin, particularly if the supplement contained liver or yeast extract. Total biotin was significantly higher than free biotin in one supplement; the difference was attributable to release of biotin rather than a biotin analog. We conclude that biotin may be present in some vitamin and nutritional supplements not labeled as containing biotin; biotin intake might be under-estimated if the subject is receiving a nutritional supplement containing extracts of liver or yeast.