We have previously shown gonadal steroid-dependent, gender specific modulation of nociception by α2-adrenoceptors. Agonist activation of the receptor enhances its association with spinophilin that antagonizes arrestin functions both by diminishing receptor phosphorylation by G-protein-coupled receptor kinase 2 (GRK2) and by competing for receptor interactions with arrestin. Since spinophilin is highly enriched in dendritic spines, we investigated whether α2-adrenoceptor-induced antinociception as well as sex-related differences are modified in spinophilin knockout mice. We evaluated α2-adrenoceptor antinociception in a heat-evoked tail flick test in spinophilin wild type (Sp+/+) and knockout (Sp-/-) mice. Baseline tail flick latencies (TFLs) did not change between any groups. Interestingly, the α2-adrenoceptor agonist, clonidine, increased TFL in male and diestrous (low estrogen) Sp-/- as well as Sp+/+ mice; in fact, this increase in TFL was significantly higher in Sp-/- male and diestrous groups than in their Sp+/+ counterparts. This unexpected finding is consistent with enhanced α2-adrenoceptor-mediated sedation observed previously in Sp-/- mice, presumably due to accelerated endocytosis of desensitized receptors and recycling of refreshed receptors when arrestin is not competed for by spinophilin in Sp-/- mice. Despite modulation of α2-adrenoceptor effects in Sp-/- mice, sex-related differences were retained; thus, clonidine was ineffective in proestrous females (highest estrogen levels), in both Sp-/- and Sp+/+ mice, reaffirming that estrogen suppresses α2-adrenoceptor-evoked antinociception. These findings show that elimination of spinophilin enhances α2-adrenoceptor-evoked antinociception in estrogen-deprived physiological settings, suggesting a role for spinophilin to suppress these effects, and yet this enhanced response cannot overcome the absence of antinociception with elevated estrogen levels. © 2008 Elsevier B.V. All rights reserved.