Unconjugated vitamin D and its metabolites were investigated in the pollen of Pinus nigra Ar. and Pinus sylvestris L. by TLC, HPLC and competitive radiochemical determination of 25-hydroxycholecalciferol (25-OHD3). It was found that vitamin D (D2, D3) was present in the pollen in amounts about 2 micrograms/10 g and 25-OHD3, 24,25-dihydroxycholecalciferol [24,25-(OH)2D3] and 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] between 0.1 and 3 micrograms/10 g of pollen, dependent on pollen species and methods.