Agonists of the melanocortin 4 (MC4) receptor have potential pharmaceutical benefit in the treatment of obesity and sexual dysfunction. In this study, we have compared the ability of a number of peptide and nonpeptide agonists to activate a FLAG-tagged human MC4 (FMC4) receptor, as measured by both cAMP accumulation and calcium mobilization using a fluorometric imaging plate reader (FLIPR). In addition, we have analyzed the ability of these agonists to cause receptor internalization, as measured by fluorescence-activated cell sorting analysis. The endogenous agonist alpha-melanocortin-stimulating hormone (alpha-MSH) increased cAMP accumulation, calcium mobilization, and receptor internalization in a dose-dependent manner in human embryonic kidney 293 cells expressing the FMC4 receptor. The activity of the other agonists varied considerably in these assays, and overall, the potency and intrinsic activity of the agonists in the cAMP accumulation assays did not correlate with their potency or intrinsic activity in either the FLIPR or receptor internalization assays. Agonists could be clearly separated into two functional classes based on their structure. Peptide agonists beta-MSH, des-acetyl-alpha-MSH, and [Nle(4), D-Phe(7)]-alpha-melanocortin-stimulating hormone exhibited 80 to 112% of the maximal alpha-MSH response in cAMP accumulation and 62 to 96% in FLIPR assays and were able to cause 75 to 118% of receptor internalization induced by alpha-MSH. Conversely, although the nonpeptide agonists exhibited 73 to 149% of the alpha-MSH response in the cAMP accumulation assays, they were significantly impaired in the FLIPR (7-40%) and receptor internalization (-5-38%) assays. These findings demonstrate an important difference in activation and internalization of the MC4 receptor by nonpeptide versus peptide agonists and provides evidence of agonist-specific conformational states.