Melanophore-stimulating hormones (MSHs) from chum salmon cause pigment dispersion in isolated melanophores of medaka, a teleost. The in vitro medaka melanophore bioassay that responded to light with pigment dispersion and to the dark with pigment aggregation was utilized for measuring the activity of melanotropic hormones. α-MSH I was the most potent melanophore-dispersing agent tested. The minimal dose for the induction of pigment dispersion was 10 −15 Mα-MSH I, 10 −13 M N-des-acetyl(Ac)-α-MSH, and 10 −11 Mβ-MSH I, respectively. The melanosome-dispersing activity of β-MSH I was enhanced about 40% by salmon N-acetyl-endorphin I ( N-Ac-EP). The results suggest that N-Ac-EP may act as an enhancer for the activity of certain MSHs. The present bioassay provides a unique method for determining the biological activity of melanotropic peptides.