We recently reported that sequential administration of estrogen and progesterone and subsequent withdrawal of progesterone increased the level of hypothalamic oxytocin (OT) messenger RNA (mRNA) in the female rat. Both estrogen priming and progesterone withdrawal are critical components of this regimen. Rats experience this ovarian steroid pattern during certain lactational events such as on days 10-12 of lactation or with interruption of nursing for 48-72 h. In the present study, we used Northern blot and in situ hybridization to determine the association between the steroid exposure and the level of OT mRNA during these lactational conditions. Week 1 lactating rats that had pups removed for 24 and 48 h, but not 12 h, had significantly higher serum estradiol concentrations than animals continuously nursing their pups on a comparable day of lactation (F4, 15 = 6; P < 0.005). Serum progesterone levels declined significantly during the 48 h after litter removal (F4, 15 = 130.5; P < 0.0001). Significant increases in OT mRNA levels were found at 48 h, but not 12 or 24 h, after litter removal (F4, 15 = 4.3; P < 0.02). Implantation of progesterone-filled capsules to compensate for the spontaneous decline in progesterone that occurs with interruption of nursing attenuated this increase in OT mRNA levels. OT mRNA was significantly higher in the hypothalamic paraventricular nuclei (P < 0.0002) and supraoptic nuclei (P < 0.002) of nursing interrupted animals receiving empty vs. progesterone-filled implants. Implantation of day 12 lactating rats with progesterone capsules for 5 days before being killed blunted the increase in OT mRNA that normally occurs on this day of lactation. The data highlight the pivotal role of estrogen priming and progesterone withdrawal in the increased expression of the OT gene during select lactational events.