Previous studies have shown that specific binding of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) to enterocyte basolateral membranes (BLM), as well as circulating concentrations, is affected in response to changes in environmental salinity. It is not known if the production of 1,25(OH)2D3 and 24,25(OH)2D3 is affected by environmental salinity. The aim of the present study was to measure the in vitro production of [3H]-1,25(OH)2D3 and [3H]-24,25(OH)2D3 in fresh water (FW) and after 1, 2, 3, and 7 days after transfer to seawater (SW). Pooled sub-cellular fractions (mitochondria and microsomes) from liver or kidney was incubated with [3H]-25(OH)D3 and the produced metabolites were separated using HPLC. Hepatic production of [3H]-1,25(OH)2D3 was decreased after 24h in SW. This was followed by an up-regulation after 48h and a second, slower decrease in production rate which leveled out after 7 days in SW. The production rate in SW was lower than the original rate in FW-adapted fish. For hepatic [3H]-24,25(OH)2D3 production the pattern was reversed. Renal production of [3H]-24,25(OH)2D3 increased significantly during the period of SW acclimation. These results suggest that environmental salinity regulates the production rate of the two antagonizing calcium regulatory hormones; 1,25(OH)2D3 and 24,25(OH)2D3. This gives further evidence to the hypothesis that there is a physiological regulation and a differentiated importance of 1,25(OH)2D3 and 24,25(OH)2D3 in relation to environmental calcium concentrations.