The rat uterus receives an innervation from the lumbosacral and thoracolumbar segments of the spinal cord. These segments receive descending oxytocinergic projections from the paraventricular nucleus of the hypothalamus. We tested the hypothesis that oxytocin regulates uterine motility through a spinal site of action. Oxytocin was administered in anesthetized female rats, either intrathecally at the lumbosacral or thoracolumbar spinal cord levels, or intravenously. Uterine activity was revealed by measuring changes of intrauterine pressure using an indwelling balloon placed in one caudal uterine horn. The uterus displayed a spontaneous activity characterized by intrauterine pressure rises, which frequency, amplitude and duration were dependent on the stage of the estrous cycle. Oxytocin delivered at the lumbosacral level affected the frequency (during proestrus, estrus and diestrus) and amplitude (during proestrus and estrus) of uterine activity. During estrus, oxytocin delivered at the thoracolumbar level affected the frequency, amplitude and duration of the intrauterine pressure rises. Intraveinous oxytocin not only affected intrauterine pressure rises (namely amplitude during proestrus and estrus, and frequency and duration during estrus) but also increased the basal tone during estrus. The effects of lumbosacral oxytocin were partly mimicked by the oxytocin agonist [Thr⁴,Gly⁷]-oxytocin, blocked by the oxytocin receptor antagonist atosiban and by hexamethonium. Arginine vasopressin delivered at the lumbosacral level had no effect. These results support our hypothesis that oxytocin, released by descending paraventriculo-spinal pathways and acting on spinal oxytocin receptors, modulates the activity of the uterus. This regulation is cycle-dependent.