The molecular recognition theory predicts that a reversed (3'→5') reading of an mRNA should yield a peptide that is structurally and functionally similar to that specified in the 5'→3' direction. We tested this idea by synthesizing a corticotropin (ACTH) analogue using a reverse reading of bovine mRNA for ACTH-(1-24). This peptide, designated ACTH-3'→5', had a similar hydropathic profile to native ACTH-5'→3' but had only 30% sequence homology and eight different charge substitutions. ACTH-3'→5' specifically bound to the surface of mouse Y-1 adrenal cells and to polyclonal anti-ACTH antibody. Additionally, ACTH-3'→5' stimulated cAMP synthesis and steroidogenesis in adrenal cells. These findings show that ACTH-3'→5' mimics the corticotropic properties of native ACTH, thereby further validating the molecular recognition theory.