Sex-related differences in antinociception produced by the activation of α 2 -adrenoceptors (α 2 -ARs) have been reported, however, the precise role of gonadal steroids is still unknown. Hence, we hypothesized that estrogen and testosterone modulate antinociceptive effects of clonidine (an α 2 -AR agonist) on N-methyl-D-aspartate (NMDA)-and heat-induced spinal nociception. We also investigated whether estrogen-or testosterone alter the expression of α 2A -adrenoceptors in the spinal cord. Sprague-Dawley (SD) rats were implanted with PE10 cannulae in the intrathecal space of the lumbosacral spinal cord and divided into male, proestrous and diestrous female, ovariectomized (OVX), estradiol-treated OVX (OVX+E), castrated male (GDX), testosterone (GDX+T) and estrogen-treated GDX (GDX+E) groups. Clonidine dose-dependently inhibited NMDA-induced scratching behavior in the male and OVX groups but to a significantly lesser extent in the OVX+E group. It also increased the tail withdrawal latency in the male, OVX, diestrous and GDX+T groups but not in the OVX+E, proestrous, GDX and GDX+E groups. Levels of α 2A -AR mRNA were significantly higher in the OVX, estradiol-treated OVX, GDX and GDX+E animals. In contrast, α 2A -AR protein levels were higher in estradiol-treated OVX, GDX, GDX+T and GDX+E animals as compared to the male. Indeed, no correlations were observed between changes in the mRNA or protein levels of α 2A -AR and behavioral observations. These results support our hypothesis that sex-related differences in α 2 -AR-mediated modulation of spinal nociception are gonadal hormone-dependent: estrogen attenuates antinociceptive effects in females whereas testosterone is required for the expression of antinociception in males. In addition, results also revealed that the mechanism of action of gonadal hormones may not involve a global alternation in expression of α 2A -AR in the spinal cord. Estrogen-induced attenuation of α 2 -AR-mediated inhibition of nociception could contribute to the higher prevalence of pain syndromes in women. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Sex-related differences in the perception of pain and the response to analgesics have been reported with females having lower thresholds for pain (Notermans, 1966;Neri et al., 1984;Berkley, 1997;Riley et al., 1999;Rollman et al., 2000; reviewed in Keogh et al., 2002) and a larger number of pain sites as compared to males (Fillingim and Maixner, 1995;Berkley, 1997;Fillingim et al., 1999). In addition, many pain syndromes/disorders such as fibromyalgia, rheumatoid arthritis, irritable bowel syndr...