We hypothesized that nitric oxide synthase (NOS) isoforms may be regulated by dynamin (DNM) in the inner medullary collecting duct (IMCD). The aims of this study were to determine which DNM isoforms (DNM1, DNM2, DNM3) are expressed in renal IMCDs, whether DNM interacts with NOS, whether a high-salt diet alters the interaction of DNM and NOS, and whether DNM activates NO production. DNM2 and DNM3 are highly expressed in the rat IMCD, while DNM1 is localized outside of the IMCD. We found that DNM1 interacts with NOS1α, NOS1β, and NOS3 in the inner medulla of male Sprague-Dawley rats on a 0.4% salt diet. DNM2 interacts with NOS1α, while DNM3 interacts with both NOS1α and NOS1β. DNM2 and DNM3 do not interact with NOS3 in the rat inner medulla. We did not observe any change in the DNM/NOS interactions with rats on a 4% salt diet after 7 days. Furthermore, NOS1α interacts with DNM2 in mIMCD3 and COS7 cells transfected with NOS1α and DNM2-GFP constructs and the NOS1 reductase domain is necessary for the interaction. Finally, COS7 cells expressing NOS1α or NOS1α/DNM2-GFP had significantly higher nitrite production compared with DNM2-GFP only. Nitrite production was blocked by the DNM inhibitor dynasore or the dominant negative DNM2K44A. Ionomycin stimulation further increased nitrite production in the NOS1α/DNM2-GFP cells compared with NOS1α only. In conclusion, DNM and NOS1 interact in the rat renal IMCD and this interaction leads to increased NO production, which may influence NO production in the renal medulla. © 2011 the American Physiological Society.