The mouse adrenocortical cell line Y1, that expresses ACTH receptors (MC2R), was used to probe the binding of ACTH and MSH peptides by using radio-labelled ACTH (1-39). The Y1 cells were found to bind [ 1 2 5 I]-labelled ACTH(1-39) with high affinity (K d 130 pM). However, none of the melanocortin peptides NDP-MSH, α-MSH, β-MSH or γ1-MSH could compete with the binding of the labelled ACTH(1-39). When other MC receptor subtype DNAs (MC1, MC3 and MC4) were transfected into the Y1 cells, characteristic binding of the [ 1 2 5 I]NDP-MSH appeared for each of the receptor subtype, but no specific binding was present in non-transfected cells. This is the first report clearly demonstrating that the ACTH receptor binds only ACTH, but not other melanocortin peptides.