We investigated the in vitro metabolism of [26,27-3H]-25-(OH)D3 in different eel tissues. After incubation with [3H]-25-(OH)D3, tissues were extracted with methanol-chloroform and chromatographed on Sephadex LH 20 columns. Two derivatives less polar than 25-(OH)D3 were detected, the first one being sensitive to KOH treatment. Three peaks more polar than 25-(OH)D3 were also found: peak I migrated close to the 24,25-(OH)2D3 area and was quantitatively the most important, but the presence of 24,25-(OH)2D3 could not be demonstrated; peak II migrated in the 1,25-(OH)2D3 region; and peak III had an elution position twice that of peak II. After 6-h incubation of tissues isolated from control eels, peak I was found in all tissues including intestine and gills. It was highest in pituitary gland and brain and lowest in ovaries and muscle. It was not significantly modified 20 days after ablation of the corpuscules of Stannius. In contrast, in vivo daily calcium chloride injection was followed 24 hr later by a significant increase in the [3H]-25-(OH)D3 conversion into peak I in gills, intestine, and the spinal cord and by an inhibition of this conversion in pituitary gland, skin, and muscle. The inhibition was found in all tissues after five daily calcium injections. Calcium injection had no effect on the in vitro metabolite synthesis by the corpuscules of Stannius. These results suggest that vitamin D is not metabolized in the same way in eel as in mammals and that this metabolism could in part be calcium dependent.