We tested for differences in aspects of mineral metabolism during the administration of diets with only vitamin D3 or only vitamin D2 in four nonhuman anthropoid primate species [two catarrhini, Macaca fascicularis (crab-eating macaque) and Macaca mulatta (rhesus macaque), and two platyrrhini, Saimiri sciureus (squirrel monkey) and Aotus vociferans (night monkey)]. All four species maintained approximately 2- to 3-fold higher serum 25-hydroxyvitamin D (25OHD) level while receiving vitamin D3 than while receiving similar amounts of vitamin D2. Serum 25OHD in M. mulatta receiving the standard primate dietary supplement of vitamin D3 was high enough (360 +/- 60 vs. 70 +/- 25 nM in vitamin D-supplemented humans; P less than 0.0001) to suggest that this widely used level of vitamin D3 supplementation is excessive for some M. mulatta. Serum 24,25-dihydroxyvitamin D [24,25-(OH)2D] in A. vociferans was uniquely high [P less than 0.01; species mean, 19 +/- 5, 95 +/- 12, and 27 +/- 5 nM in groups receiving diets with 1.5 IU vitamin D3/g, 6.6 IU vitamin D3/g, and 15 IU vitamin D2/g, respectively; mean 24,25-(OH)2D from the other three species pooled across three diets was 7 +/- 5 nM]. We confirmed relative resistance to 1,25-(OH)2D in S. sciureus, manifested by osteomalacia and moderately high serum 1,25-(OH)2D. Serum 1,25-(OH)2D in S. sciureus increased 4-fold (P less than 0.05) when the precursor in serum was changed from 250HD3 to 250HD2, suggesting that this species shows more severe resistance to 1,25-(OH)2D2 than to 1,25-(OH)2D3. In conclusion, we found many differences in vitamin D metabolism among four nonhuman anthropoid primate species. The striking feature in A. vociferans (high, 24,25-(OH)2D without high 25OHD in serum independent of whether diet contained only vitamin D3 or only vitamin D2) should allow determination of whether 24,25-(OH)2D functions as a unique agonist or an inactive metabolite in this species.