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Intermediate and anterior lobes from the pituitary glands of female Wistar rats were freshly dissected and chromatographed on Sephedex G-50 and BioGel P6. Fractions were monitored with radioimmunoassays for NH₂- and CO₂H-terminal adrenocorticotrophin (ACTH), -melanocyte-stimulating hormone (-MSH), and - and β-endorphin. A large molecular weight, glycosylated form of corticotrophin-like intermediate lobe peptide (CLIP) which we have termed big CLIP, and a β-MSH-like molecule were identified in the pars intermedia and in both lobes, a major peak of activity with the elution characteristics and cross-reactivity of -lipotrophic hormone (-LPH) was detected. In the pars distalis, the larger peptides 1–39 ACTH and β-LPH predominated, whereas in the pars intermedia, the smaller peptides -MSH, CLIP, β- and -endorphin were more abundant. Chromatography of rat plasma revealed peaks of immunoreactivity in the corresponding positions to those detected in the pituitary gland.