The present investigation was carried out to study the conversion of [3H]vitamin D3 and 25-OH-D3 in vivo and in vitro in freshwater tilapia (Oreochromis mossambicus). A metabolite co-migrating with 1,25-(OH)2D3 formed from [3H]vitamin D3 in vivo was about 3-fold more concentrated than that of 25-OH-D3. The conversion of 25-OH-[3H]D3 to a product co-eluting with 1,25-(OH)2D3 was several-fold higher in liver than in kidney. Our data suggests that the tilapia 'liver' may be a major site for vitamin D metabolism.