The receptor for melanin-concentrating hormone (MCH) was recently identified as the orphan G protein-coupled receptor SLC-1. In this study, a CHO cell line expressing the MCH receptor (K_d= 1.3 nm; binding capacity, 3.6 pmol/mg protein) is used to assess the ability of the MCH receptor to couple to G_i, G_o, and G_q proteins. The results demonstrate that MCH inhibits forskolin-stimulated cAMP production in a pertussis toxin- (PTX)-sensitive manner in CHO-MCHR cells (EC₅₀= 100 pm), indicating that the MCH receptor couples to one or more members of the G_i subfamily of G proteins. In addition, MCH stimulates increases in phosphoinositide metabolism (EC₅₀ = 50 nm) and in intracellular free Ca²⁺ levels (EC₅₀ = 10 nm). MCH-stimulated inositol phosphate production and increases in intracellular free Ca²⁺ are partially inhibited (60% and 40%, respectively) by PTX pretreatment, demonstrating that there are at least two components of each of these signaling pathways. One component is PTX sensitive and therefore mediated through a G_i/G_o protein. A distinct G protein-coupled (probably G_q type) mediates the PTX-insensitive component. To distinguish G_ivs. G_o coupling, MCH-stimulated mitogen-activated protein (MAP) kinase activity was examined. G_i and G_o use separate signaling pathways to mediate MAP kinase activation in CHO cells. Protein kinase C (PKC) activity is essential in the G_o-dependent MAP kinase signaling pathway, but is not required in the G_i-dependent MAP kinase signaling pathway. MCH stimulated MAP kinase activity is decreased (50%), but not abolished, by inhibition of PKC activity or depletion of cellular PKC, indicating that MCH-stimulated MAP kinase activity is mediated through both G_i- and G_o-dependent signaling mechanisms. The results of this study are the first to clearly demonstrate that the MCH receptor couples to multiple G proteins to mediate several diverse intracellular signaling pathways.