The expression of preproenkephalin (PPE) mRNA has previously been shown to be regulated by steroid hormones in the ventromedial nucleus of the hypothalamus (VMH) and to be regulated by noxious stimuli in the dorsal horn of the spinal cord (DH). The present in situ hybridization study in ovariectomized rats showed that PPE mRNA expression in both the VMH and the lumbar DH, responds to the interaction between a noxious peripheral stimulus and ovarian steroid hormones. In the VMH, either estradiol or estradiol + progesterone increased the mean PPE mRNA content per cell by 100% compared with vehicle-treated rats. Unilateral hindpaw injection of 5% formalin, as compared to saline, significantly increased mean PPE mRNA content per VMH cell in rats treated with vehicle or estradiol but not those treated with estradiol + progesterone. Regression analysis for mean PPE mRNA content per VMH cell as a function of intensity of hindpaw inflammation showed a significant positive correlation coefficient after vehicle and estradiol treatment (P < 0.02) but a strong trend towards a negative correlation coefficient after estradiol + progesterone treatment (P < 0.06). ANOVA for homogeneity of regression coefficients showed a significant difference across hormone groups (P < 0.01). In the lumbar DH, mean PPE mRNA content per cell was greater in rats injected with formalin than with saline and was greatest in rats given steroids + formalin. Mean PPE mRNA content per DH cell was greater ipsilateral than contralateral to the formalin injection in estradiol-treated rats, but no laterality difference was seen in the other hormone groups. No significant differences in mean PPE mRNA levels per DH cell were found among the rats treated with saline + hormone, saline + vehicle, formalin + vehicle, or uninjected rats. For all hormone groups combined, mean PPE mRNA per DH cell showed a significant positive regression on intensity of hindpaw inflammation (P < 0.05). Taken together these data are consistent with reports of increased pain threshold during pregnancy, descending control of antinociception from the basomedial hypothalamus and positive correlations between VMH levels of PPE mRNA and lordosis, a behavior evoked by somatosensory stimulation below nociceptive threshold.