A sensitive in situ melanin assay using cultured mouse B16 melanoma cells is described for structure-activity studies with melanocyte-stimulating hormone (MSH) peptides. B16 Cells were seeded at a density of 2500 cells per well in 96-well microtest tissue culture plates; after 24 h the cells were incubated in the presence of serial dilutions of MSH peptides for 3 to 5 days. The melanin released into the medium of each well was then determined spectrophotometrically at a wavelength of 405 nm using an automatic microplate reader calibrated against synthetic melanin. Studies with alpha-MSH, [Nle4, D-Phe7]-alpha-MSH, [3'-iodo-Tyr2]-alpha-MSH, adrenocorticotropin (ACTH)(1-24), and ACTH(1-39) showed that the peptides had identical intrinsic activities and that the relative potencies were similar to those obtained with a tyrosinase assay. The EC50 of alpha-MSH was 27 pM, i.e., about five- to sevenfold lower than that in the assays for tyrosinase or intracellular melanin. Thus, the new assay represents the most sensitive melanoma cell assay for MSH available to date.