Oxytocin (OT) is essential for parturition and milk ejection, and OT-containing fibers are present in several regions of the brain and in the spinal cord. During lactation, activation of spinal cord neurons by suckling stimulation involves deep laminae III-X including sympathetic preganglionic neurons of the intermedio-medial cell column. In the present study, experiments were designed to determine if the suckling provided by the litter increased OT levels in the spinal cord of dams, as determined by competitive immunoassay. In addition, we investigated if OT fibers reach neurons of the spinal cord that are known to respond to suckling. The OT content was higher in the hypothalamus than in the spinal cord in animals from all experimental groups. After 6 h of pup separation, OT levels decreased and suckling for 5 min induced a significant increase of OT levels in the spinal cord. Double immunostaining for Fos and OT showed OT-positive fibers adjacent to neurons that had Fos-positive nuclei, located mostly in laminae III, IV, and X. The present data support the notion that OT is released within the spinal cord in response to suckling, suggesting a role for this peptide in modulating the afferent and/or efferent responses generated by suckling.