Abstract Previous studies in female rats have shown that estrogen increases preproenkephalin (PPE) mRNA levels in the ventrolateral part of the ventromedial nucleus of the hypothalamus (VMHVL), an area implicated in the modulation of sexual behavior. In order to assess the physiological role of hypothalamic opioid expression in lordosis reflex 16-mer oligodeoxynucleotide (ODN) directed towards the PPE mRNA were acutely microinjected above the VMH of estradiol-primed ovariectomized rats. Estradiol-induced lordosis behavior was observed in response to a stud male 2 days thereafter. Antisense (without or with 4 mismatches) ODN injections near the VMHVL resulted in a significant reduction in lordosis quotient compared to control (reverse sense) ODN treatment or to antisense ODN injections targeted anterior or posterior to the VMHVL. In contrast, locomotor activity of these animals in the open-field test was not affected by ODN treatments. Enkephalin immunoreactive levels were determined by radioimmunoassay in the preoptic area, a major terminal field of the VMHVL. Estradiol-induced enkephalin levels were greatly reduced in antisense-treated groups. Using the in situ hybridization technique, PPE mRNA levels in the VMHVL were also determined. A 1.5–2-fold increase in PPE mRNA levels was observed in estradiol-treated rats compared to ovariectomized rats as previously described. This increase in PPE mRNA levels was not affected by ODN treatment, suggesting that the reduction of enkephalin expression was mainly due to physical blockade of PPE mRNA translation and not to its degradation. Taken together, these data further support the behavioral role of PPE expressing VMHVL neurons. They also highlight the in vivo potency of acute administration of antisense phosphorothioate ODNs in blocking neuronal target gene expression.