1. An in vitro bioassay for melanotropic peptide utilizing reflectance measurements of toad skin (Bufo ictericus ictericus) is described as an alternative to the commonly used Rana pipiens bioassay. 2. The toad skin bioassay is as sensitive to melanotropins and melanin concentrating hormone (MCH) as the frog bioassay. 3. On the basis of parallel dose-response curves obtained with the toad skin assay we found that beta-MSH is slightly less active than alpha-MSH, whereas the synthetic analogue [Nle4-D-Phe7]-alpha-MSH is about 10 times more potent and exhibits prolonged biological activity. 4. MCH, a putative neurohormone, can also be bioassayed in the in vitro toad skin bioassay, since it has alpha-MSH-like activity on amphibian melanocytes. 5. Since neither adreno- nor cholinoceptors are present in the toad melanocytes, the assay provides great specificity and sensitivity for the determination of melanotropin activity in tissue or blood.