BACKGROUND Sex differences in pain have been well documented; however, the mechanisms involved remain to be elucidated. The present study examined whether sex differences exist in the functioning of primary afferent fibres by assessing formalin-evoked release of substance P by way of neurokinin 1 receptor (NK1r) internalization. The study also investigated whether the observed effects would be oestradiol-sensitive. METHODS Intact and gonadectomized male and female rats were given intraplantar formalin and then euthanized either 5 or 30 min later, representing phase 1 or 2 of the formalin response, respectively. In a subsequent experiment, ovariectomized females received oestradiol prior to formalin administration. Lastly, formalin-evoked NK1r internalization was assessed across the female oestrous cycle. RESULTS Intraplantar formalin evoked significant NK1r internalization, during phase 1 and 2, in both males and females. During phase 1, no differences in NK1r internalization were detected between males or females, regardless of the gonadal status. In contrast, during phase 2, intact females exhibited greater NK1r internalization than intact males. Moreover, ovariectomy reduced NK1r internalization as compared to intact females, whereas castration had no effect as compared to intact males. Oestradiol supplementation in ovariectomized females increased NK1r internalization to levels observed in intact females. Formalin-evoked NK1r internalization did not differ across the oestrous cycle. CONCLUSIONS These findings suggest that oestradiol mediates sex differences in formalin-evoked substance P release, which may contribute to a differential development of central sensitization and pain behaviours in males and females.