The agouti gene codes for agouti signaling protein (ASP), which is temporally expressed in wild-type mouse follicular melanocytes where it induces pheomelanin synthesis. Studies using purified full-length agouti signaling protein has shown that it competes with (&agr;)-melanocyte stimulating hormone for binding to the melanocortin 1 receptor. We have investigated whether ASP binds exclusively to the melanocortin 1 receptor expressed on mouse melanocytes in primary culture, or additionally activates a receptor that has not been identified yet. We have compared the responses of congenic mouse melanocytes derived from C57 BL/6J-E(+)/E(+), e/e, or E(so)/E(so) mice to (alpha)-MSH and/or ASP. E(+)/E(+) melanocytes express the wild-type melanocortin 1 receptor, e/e melanocytes express a loss-of-function mutation in the melanocortin 1 receptor that results in a yellow coat color, and E(so)/E(so) is a mutation that causes constitutive activation of the melanocortin 1 receptor and renders melanocytes unresponsive to (alpha)-melanocyte stimulating hormone. Mouse E(+)/E(+) melanocytes, but not e/e or E(so)/E(so) melanocytes, respond to agouti signaling protein with decreased basal tyrosinase activity, and reduction in levels of tyrosinase and tyrosinase-related proteins 1 and 2. Only in E(+)/E(+) melanocytes does agouti signaling protein abrogate the stimulatory effects of (alpha)-melanocyte stimulating hormone on cAMP formation and tyrosinase activity. These results indicate that a functional melanocortin 1 receptor is obligatory for the response of mammalian melanocytes to agouti signaling protein.