1. The aims of this study were: (i) to characterize the subcellular localization of nitric oxide synthase (NOS) 1 and NOS3 activity and expression within the cytosolic, plasma membrane and intracellular membrane subcellular fractions of the renal inner medulla of rats; and (ii) to determine whether NOS1 and NOS3 activity and expression in subcellular fractions of the renal inner medulla are regulated by dietary salt intake. Although the NOS system is important in maintaining Na+ and water homeostasis, the identity of the NOS isoform that is sensitive to dietary Na+ remains unclear. In addition, subcellular localization of both NOS1 and NOS3 has been shown to regulate enzymatic activity and influence the ability of NOS to produce nitric oxide (NO). 2. Renal inner medullae were dissected from male Sprague-Dawley rats and separated into cytosolic, plasma membrane and intracellular membrane fractions for measurement of NOS activity and western blot analysis. 3. On a normal-salt diet, NOS activity and NOS1 and NOS3 protein expression were present in all three subcellular fractions, although total NOS activity was enriched in the intracellular membrane fraction. In response to a high-salt diet, urinary nitrate/nitrite (NOx) increased. Despite an increase in NO x excretion, total NOS activity in the renal inner medullary homogenate was decreased. There were no detectable differences in NOS activity in the subcellular fractions. Expression of NOS1 protein was decreased in the cytoplasmic and plasma membrane fractions, although maintained in the intracellular membrane fraction, in response to high salt. Expression of NOS3 protein was unaffected by high salt. 4. In conclusion, we hypothesize that NOS1 localization in the intracellular membrane is important in increasing NO production to aid Na+ and water homeostasis. © 2007 The Authors.