Mammalian type-II sodium-phosphate cotransporter (NaPi-II) and inorganic phosphate uptake stimulator (PiUS) genes are upregulated by dietary phosphorus (P) restriction to increase intestinal and renal P transport. Little is known, however, about sites of expression of NaPi-II and PiUS or about dietary regulation of P transport in other vertebrates. We therefore studied the tissue distribution and dietary regulation of NaPi-II, PiUS and sodium-glucose cotransporter (SGLT1, negative control) mRNA as well as of NaPi-II protein in juvenile rainbow trout (Oncorhynchus mykiss). We also determined both chronic and acute effects of dietary P on intestinal Pi absorption in vivo. NaPi-II, PiUS, and SGLT1 mRNA were all found in the proximal and distal intestine, pyloric caeca, and kidney. PiUS mRNA was also found in the heart, gill, blood, stomach, liver, skin, and muscle. The tissue distribution of NaPi-II protein correlated with that of NaPi-II mRNA. However, NaPi-II antibodies also recognized related epitopes in gill ionocytes not expressing NaPi-II mRNA. Chronic consumption of a low P diet increased NaPi-II and, to a lesser extent, PiUS but not SGLT1 mRNA abundance in the intestine and kidney. Unlike mammals, there was no detectable shift in tissue or cellular localization of renal and intestinal NaPi-II protein in response to dietary P restriction. Regulation of NaPi and PiUS mRNA expression was observed only in fish grown under optimal aqueous oxygen concentrations, suggesting that dietary regulation itself can be influenced by environmental factors. In vivo fractional absorption of Pi by the intestine decreased in fish fed high P diets. Decreases in absorption were less pronounced in fish previously fed low P diets, suggesting that P status (diet history) modulates acute regulation of P absorption. One of the mechanisms underlying the regulation of dietary Pi absorption in vivo may be a specific change in intestinal NaPi-II and PiUS gene expression.