Rat spinal cord contains the opioid peptide including [Met5]enkephalin-Arg6-Gly7-Leu8 (YGGFMRGL) and a higher molecular weight (HMW) immunoreactive peptide which is an N-terminal extended molecular form of YGGFMRGL. Since a high proportion of tissue immunoreactivity resides in the HMW component we have determined whether this form is released during perfusion of the spinal cord subarachnoid space in vivo while (1) electrically stimulating the sciatic nerves bilaterally or (2) superfusing with substance P. We have found that YGGFMRGL and the HMW immunoreactivity are released by both types of stimuli. The HMW material appeared to be the more stable of the two species of immunoreactivity; its presence in the superfusate was more consistently observed than that of YGGFMRGL itself. Injection of YGGFMRGL into the spinal subarachnoid space in chronically catheterized rats produced a suppression of the tail-flick response. This effect of YGGFMRGL was reversed by naloxone suggesting an action mediated by spinal opiate receptors. These data suggest that YGGFMRGL plays an integral role in the neurotransmission process between spinal neurons storing enkephalin and other neurons. The possibility that enkephalin-mediated neurotransmission includes multiple chemical signals can be entertained.