This chapter discusses the relationship between the three-dimensional conformation and biological activity of α-melanophore-stimulating hormone (α-MSH). The three-dimensional conformation and biological activity in α-MSH has been examined by a variety of researchers using numerous biophysical methods including reversed phase high-performance liquid chromatography, circular dichroism, computer-assisted molecular modeling, proton and carbon-13 nuclear magnetic resonance spectroscopy. Biologically, a-MSH is primarily known for its ability to stimulate integumental melanocytes, but it has also been implicated in a variety of other physiological and neurological processes. Reversed phase high performance liquid chromatography has found significant application in the purification of biologically active compounds, such as peptides and proteins. The ability of circular dichroism to provide critical insight into the conformations of small linear peptides is still debated. A few studies of α-MSH and its linear and cyclic analogs have been carried out in an effort to understand the bioactive conformation of this peptide hormone.