The DNA encoding the human melanocortin 4 receptor was expressed in COS (CV-1 origin, SV 40) cells and its radioligand binding properties was tested by using the [125I][Nle4, D-Phe7] alpha-melanocyte stimulating hormone (MSH). The radioligand was found to bind to a single saturable site with a Kd of 3.84 +/- 0.57 nmol/l in the MC4 receptor expressing cells. The order of potency of a number of substance competing for the [125I][Nle4, D-Phe7] alpha-MSH binding was the following; [Nle4, D-Phe7] alpha-MSH > [Nle4]-alpha-MSH > beta-MSH > desacetyl-alpha-MSH > alpha-MSH > ACTH (1-39) > ACTH (4-10) > gamma 1-MSH > gamma 2-MSH. This order of potency is unique for the melanocortin 4 receptor when compared to our previously published data for the other melanocortin receptor subtypes. Most notably the melanocortin 4 receptor shows highest affinity for beta-MSH, among the endogenous MSH-peptides. Furthermore the melanocortin 4 receptor shows very low affinity for the gamma-MSH peptides. This distinguishes the melanocortin 4 receptor from the melanocortin 3 receptor, which is the other major central nervous system melanocortin-receptor, as melanocortin 3 receptor shows high affinity for gamma-MSH. Our finding might indicate a specific role for beta-MSH for the melanocortin 4 receptor.