The ghrelin receptor [growth hormone secretagogue receptor (GHSR)-1a] represents a promising pharmacologic target for the treatment of metabolic disorders, including obesity and cachexia, via central appetite modulation. The GHSR-1a has a complex pharmacology, highlighted by G-protein-dependent and -independent downstream signaling pathways and high basal constitutive activity. The functional selectivity and signaling bias of many GHSR-1a-specific ligands has not been fully characterized. In this study, we investigated the pharmacologic properties of ghrelin, MK-0677, L692,585, and [d-Lys3]-growth hormone-releasing peptide-6 (Dlys), JMV2959, and [d-Arg(1),d-Phe(5),d-Trp(7, 9),Leu(11)]-substance P (SP-analog). We investigated their effect on basal GHSR-1a constitutive signaling, ligand-directed downstream GHSR-1a signaling, functional selectivity, and signaling bias. Dlys behaved as a partial antagonist with a strong bias toward GHSR-1a--arrestin signaling, whereas JMV2959 acted as a full unbiased GHSR-1a antagonist. Moreover, the SP-analog behaved as an inverse agonist increasing G-protein-dependent signaling, but only at high concentrations, whereas, at low concentrations, the SP-analog attenuated -arrestin-dependent signaling. Considering the limited success in the clinical development of GHSR-1a-targeted drugs so far, these findings provide a novel insight into the pharmacologic characteristics of GHSR-1a ligands and their signaling bias, which has important implications in the design of novel, more selective GHSR-1a ligands with predictable functional outcome and selectivity for preclinical and clinical drug development.Ramirez, V. T., van Oeffelen, W. E. P. A., Torres-Fuentes, C., Chrucicka, B., Druelle, C., Golubeva, A. V., van de Wouw, M., Dinan, T. G., Cryan, J. F., Schellekens, H. Differential functional selectivity and downstream signaling bias of ghrelin receptor antagonists and inverse agonists.