Biochemical and pharmacological studies have suggested that NOS2 (inducible nitric oxide synthase) has a functional role in the blood pressure response to increases in dietary salt intake. On a high-salt diet, the Dahl/Rapp salt-sensitive (S) strain of rat, a genetic model of salt-sensitive hypertension, did not show increased nitric oxide production. NOS2 from S rats possesses a point mutation that results in substitution of proline for serine at position 714. In the present study, rat NOS2 was shown to be ubiquitinated in vitro and in vivo and to be degraded by the proteasome; this process was accelerated for the S714P mutant. Accelerated degradation of the S714P mutant enzyme accounted for the diminished enzyme activity of this mutant. Hsp90 (heat-shock protein 90) associated with NOS2 and modulated degradation, but was not responsible for the accentuated degradation of the S714P mutant enzyme. The combined findings demonstrate the integral role of ubiquitination and degradation by the proteasome in the regulation of NO production by rat NOS2. Demonstrating that this process is responsible for the abnormal function of the S714P mutant NOS2 in S rats confirms the physiological importance of the proteasome in NOS2 function.