Melanin-concentrating hormone (MCH), an orexigenic neuropeptide in mammals, activates a G-protein coupled receptor, MCHR1. It is expected that antagonists of MCHR1 function will prove therapeutically useful as anti-obesity agents. Intracellular signaling by MCHR1 has been investigated primarily using non-neural cell lines expressing the recombinant receptor, in which MCHR1 has been shown to couple to G alpha(i/o) and G alpha(q) G-proteins. While these cell lines have been widely utilized to discover and optimize small molecule antagonists, it is unknown whether the intracellular signaling pathways in these cells accurately reflect those in neurons. Thus, we sought to develop a neurally derived cell line endogenously expressing MCHR1. IMR32, a human neuroblastoma cell line, has been shown to express MCHR1 mRNA; however, we were unable to detect either MCH-binding or MCH-stimulated Ca++-mobilization in these cells. Following transfection of IMR32 cells with a plasmid encoding human G alpha(16) G-protein, we isolated a cell line, I3.4.2, which responded to MCH in Ca++-mobilization assays. We found that the expression level of MCHR1 mRNA in I3.4.2 cells was 2000-fold higher than in the parent cell line. Using [125I]MCH saturation-binding to I3.4.2 cell membranes, we estimated the Bmax as 0.72 pmol/mg protein and the Kd as 0.35 nM. We report that Ca++-mobilization in I3.4.2 cells was insensitive to pertussis toxin (Ptx) treatment, indicating that signaling was via G alpha(q) G-proteins. Furthermore, negative results in cAMP accumulation assays confirmed the lack of signaling via the G alpha(i/o) G-proteins. Our results suggest that the I3.4.2 cell line may be useful for characterization of MCHR1 activity in a neural-derived cell line.