Following periods of microgravity or bed rest, individuals often exhibit reduced VO_2max, hypovolemia, cardiac and vascular effects, and autonomic dysfunction. Recently, alterations in the expression of vascular and central nervous system nitric oxide (NO) synthase have been observed in hindlimb unloaded (HU) rats, a model used to simulate physiological effects of microgravity or bed rest. The present study examined the effects of 14 days of hindlimb unloading on hemodynamic responses to systemic nitric oxide synthase (NOS) inhibition in conscious control and HU rats. Since differences in NO and autonomic regulation might occur following HU, we also evaluated potential differences in resting autonomic tone and the effects of NOS inhibition after autonomic blockade. Administration of L-nitro-arginine methyl ester (L-NAME, 20 mg/kg, i.v.) increased mean arterial pressure (MAP) to similar levels in control and HU rats. However, the change in MAP to L-NAME was less in HU rats which had an elevated baseline MAP. In separate experiments, atropine (1 mg/kg, i.v.) increased heart rate (HR) in control but not HU rats. Subsequent administration of the ganglionic blocker hexamethonium (30 mg/kg, i.v.) decreased MAP and HR to a greater extent in HU rats. Administration of L-NAME following autonomic blockade increased MAP in both groups to a greater extent compared to intact conditions. However, the pressor response to L-NAME was still reduced in HU rats. These data suggest that hindlimb unloading in rats reduces peripheral nitric oxide as well as cardiac parasympathetic tone. Along with elevations in sympathetic tone, these effects likely contribute to alterations in vascular control and changes in autonomic reflex function following spaceflight or bed rest.