Vitamin D-deficient rats given an aqueous extract of the South American plant Solanum glaucophyllum accumulate 1,25-dihydroxyvitamin D3 in their blood and intestines at the time they show enhanced intestinal calcium absorption. The identity of the 1,25-dihydroxyvitamin D3 was established by co-chromatography with 1,25-dihydroxy[23,24-3H]vitamin D3 on Sephadex LH-20 columns, microparticulate silica gel columns, a reversed-phase column developed under high pressure, and by a specific 1,25-dihydroxyvitamin D3 binding assay. The chromatographic systems used are fully capable of resolving all of the known metabolites of vitamin D3. Serum of the S. glaucophyllum-treated rats showed 300 pg/ml of 1,25-dihydroxyvitamin D3 and no detectable 1,25-dihydroxyvitamin D2. Similarly, intestine of such rats had 230 pg/g of 1,25-dihydroxyvitamin D3. Control animals which received the vehicle instead of S. glaucophyllum had only 20 pg/ml of 1,25-dihydroxyvitamin D3 in their serum and 4.4 pg/g of 1,25-dihydroxyvitamin D2 in their intestine. These results demonstrate that S. glaucophyllum extracts must be a source of 1,25-dihydroxyvitamin D3; thus a significant basis for the calcinogenic properties of S. glaucophyllum must be the presence of a conjugated form of 1,25-dihydroxyvitamin D3, which is rendered available by digestion.