Previous work has shown that astronauts returning from space often experience orthostatic hypotension resulting from inadequate peripheral vasoconstriction. Using the hindlimb-unloaded (HU) rat as a model to simulate microgravity, the purpose of this study was to determine whether 14 days of HU alters vasoconstrictor responsiveness of mesenteric resistance arteries. Mesenteric resistance arteries from control (n = 43) and HU (n = 44) rats were isolated, cannulated and pressurized to 108 cm H₂O for in vitro experimentation. Myogenic (intralumenal pressure ranging from 30-180 cm H₂O), KCl (2-100 mM), norepinephrine (NE, 10^-9-10^-4 M) and caffeine (1-20 mM) induced vasoconstriction, as well as the temporal dynamics of vasoconstriction to NE, were determined. The active myogenic and passive pressure responses were unaltered by HU when pressures remained within physiological range. However, vasoconstrictor responses to KCl, NE, and caffeine were diminished by HU, as well as the rate of constriction to NE (C, 14.8 ± 3.6 µm/s vs. HU 7.6 ± 1.8 µm/s). Expression of sarcoplasmic reticulum Ca²⁺ATPase 2 and ryanodine 3 receptor mRNA were unaffected by HU, while ryanodine 2 receptor mRNA and protein expression were diminished in mesenteric arteries from HU rats. These data suggest that HU-induced and microgravity-associated orthostatic intolerance may be due in part to an attenuated vasoconstrictor responsiveness of mesenteric resistance arteries resulting from a diminished ryanodine 2 receptor Ca²⁺ release mechanism.