Bioassays have played an important role in determining the presence of hormones in the blood or other tissues as well as determining the biological potency of structural analogs of hormones. The earliest studies of melanophore responses to melanotropins utilized a so-called "melanophore index". The skin of the frog has been most widely used for the study of the melanotropic activity of peptides. Skins from the leopard frog, Rana pipiens, have been most commonly employed in the in vitro bioassay. A. J. Kastin used hypophysectomized R. pipiens and found by using the classical melanophore index that the in vivo assay was as sensitive as the in vitro assay for the determination of the potency of a number of adrenocorticotropin-like peptides. Lizard skin bioassay, although approximately one order of magnitude less sensitive than the frog skin bioassay, is particularly useful since the darkening response to melanotropins and the subsequent lightening response after their removal are very rapid events.