The single tryptophan residue in the pituitary hormone ACTH was modified selectively by reaction with a variety of substituted o-nitrophenysulfenyl chlorides. In addition to quantitative modification of the tryptophan residue, the reaction invariably resulted in partial oxidation of the methionine residue to the sulfoxide. The methionine sulfoxide derivative was separated from the desired product by partition chromatography on Sephadex G-50 in the solvent system 1-butanol-pyridine-0.1% acetic acid (5:3:11). Thus, the 2,4-dinitrophenylsulfenyl, 2-nitro-4-carboxyphenylsulfenyl and 2-nitro-4-carbamidophenylsulfenyl derivatives of ACTH were prepared and characterized. Modifications in the isolation of ACTH from ovine pituitaries are also described. The melanocyte stimulating activities of the native hormone and the analogues are discussed. The steroidogenic potency of ACTH fractions was estimated in vivo in rats; melanocyte-stimulating activity was measured in vitro in frog skin.