Bioavailability of vitamin D (D) was assessed by competitive protein-binding assay of serum 25-hydroxyvitamin D (25OHD) levels in normal adult volunteers after a single oral, sc, or im dose of commercially available D Pharmaceuticals or after a single iv injection of ethanol-propylene glycol solution containing D. Similar increases in serum 25OHD levels were noted after either iv D2 or D3 (100 μg/kg), suggesting that the 25-hydroxylation of D2 and D3 is comparable in man. Absolute increases in serum 25OHD levels were similar in subjects receiving D in iv doses of 100 and 250 μg/kg; however, subjects receiving the larger dose had higher basal 25OHD levels. This finding suggests an inverse relationship in man between basal serum 25OHD concentrations and relative serum 25OHD increments after administration of pharmacological doses of D. Oil depot sc and im injection of D (200 MgAg) resulted in delayed serum 25OHD increases compared to oral and iv dosing (100 μg/kg). Studies after im oil depot injection of [3H]D3 into rats showed that D administered in this manner had delayed bioavailability but remained unaltered in situ. Differences in D pharmaceutical bioavailability should be considered in treatment or prophylaxis with this sterol.