We have compared the affinities and anti-opioid activities of the different peptides putatively produced by the rat NPFF precursor, NPAFLFQPQRF-NH2 (:NPA-NPFF) and EFWSLAAPQRF-NH2 (EFW-NPSF), with those already identified in nervous tissue, FLFQPQRF-NH2 (NPFF) and SLAAPQRF-NH2 (NPSF), NPFF and NPA-NPFF exhibit a high affinity (0.34 and 0.14 nM, respectively) for [I-125]1DMe binding sites of the rat spinal cord. In contrast, EFW-NPSF displays an affinity 13 times higher than NPSF (1.99 and 9.5 nM, respectively). In rat dorsal raphe neurones, EFW-NPSF, NPFF, and NPA-NPFF maximally reduce the inhibitory effect of nociceptin on the [Ca2+](i) transients triggered by depolarization by 39, 31, and 58%, respectively. NPSF is inactive in the same test. We conclude that NPA-NPFF and EFW-NPSF are likely to be the physiologically active neurotransmitters in rat brain.