The alpha-hemoglobin-derived dodecapeptide RVD-hemopressin (RVDPVNFKLLSH) has been proposed to be an endogenous agonist for the cannabinoid receptor type-1 (CB1). To study this peptide we have raised monoclonal antibodies (mAbs) against its C-terminal part. Using an immunoaffinity mass spectrometry approach a whole family of N-terminally extended peptides in addition to RVD-Hpa were identified in rodent brain extracts and human and mouse plasma. We designated these peptides Pepcan-12 (RVDPVNFKLLSH) to Pepcan-23 (SALSDLHAHKLRVDPVNFKLLSH), referring to peptide length. The most abundant Pepcans found in the brain were tested for CB1 receptor binding. In the classical radioligand displacement assay, Pepcan-12 was the most potent ligand but only partially displaced both [3H]CP55,940 and [3H]WIN55,212-2. The data were fitted with the allosteric ternary complex model revealing a cooperativity factor value a>1, thus indicating a negative allosteric modulation. Dissociation kinetic studies of [3H]CP55,940 in the absence and presence of Pepcan-12 confirmed these results by showing increased dissociation rate constants induced by Pepcan-12. A fluorescently labeled Pepcan-12 analogue was synthesized to investigate the binding to CB1 receptors. Competition binding studies revealed Ki values of several Pepcans in the nM range. Accordingly, using competitive ELISA we found low nM concentrations of Pepcans in human plasma and ~100 pmol/g in mouse brain. Surprisingly, Pepcan-12 exhibited potent negative allosteric modulation (20-50%) of the orthosteric agonist-induced cAMP accumulation, [35S]GTP?S binding and on CB1 receptor internalization. Pepcans are the first endogenous allosteric modulators identified for CB1 receptors. Given their abundance in the brain, Pepcans could play an important physiological role in modulating endocannabinoid signaling.