Abstract: Binding activity and biologic effect of a novel α-melanocyte-stimulating hormone analogue were tested on cells transiently expressing the human melanocortin-1 (MC1), MC3, MC4, and MC5 receptors. The human MC1 and MC5 receptor genes were cloned into the expression vector pcDNA3.1/myc-his(-)B. The vectors were transferred to HEK-293 cells by the calcium phosphate method. Stable receptor populations were generated using G418 selection (900 μg·mL^-1) for subsequent bioassay analysis. K_i values of the novel α-MSH analogue for MC1, MC3, MC4, and MC5 receptors were obtained in competition with ［¹²⁵I］-NDP-MSH for binding studies. The cyclic AMP level was tested by using ［³H］-cyclic AMP kit. It is showed that K_i values of the novel α-MSH analogue for MC1, MC3, MC4, and MC5 receptors were(0.159±0.040), (35.430±6.743), (19.293±2.780) and (2.230±0.670) nmol·L^-1, respectively. Its EC₅₀ values for MC1, MC3, MC4, and MC5 receptors were (0.45±0.07), (7.80±0.65), (2.55±0.23) and (0.33±0.09) nmol·L^-1, respectively. In these tests, the novel α-MSH analogue is a MC1R and MC5R selective agonist.