Cholecalciferol (Vitamin D3) increased plasma inorganic phosphate concentration in American eels,Anguilla rostrata, in a dose-dependent fashion. This response was more marked in phosphate loaded fish. In control as well as phosphate loaded eels the hyperphosphatemic response to D3 was associated with a sharp reduction in renal phosphate clearance relative to(14)C-polyethelene glycol (PEG) clearance. Glomerular filtration and urine flow rates were not affected by D3. As renal phosphate clearance, even in phosphate loaded eels, never significantly exceeded that of PEG, it is suggested that D3 reduced the relative clearance rate of phosphate by increasing renal phosphate reabsorption rather than by reducing the tubular secretion of phosphate.