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Using RIAs for six regions within proopiolipomelanocortin (proOLMC), gel filtration, and electrophoresis, we studied pituitary peptides in a normal horse and one with Cushing's disease caused by a pars intermedia adenoma. Almost all immunoreactive (IR) ACTH (78%) was 4,500 mol wt (4.5K) ACTH in normal pars distalis, but it was almost 100% corticotropin-like intermediate lobe peptide (CLIP) in normal pars intermedia. alpha MSH and beta MSH were found mainly in pars intermedia: equal concentrations of the beta MSH precursors, beta-lipotropin (beta LPH) and gamma LPH, were found in pars distalis. Most IR-beta-endorphin (IR-beta END) was found as beta END in pars intermedia, but roughly equal concentrations of beta END and its precursor, beta LPH, were found in pars distalis. A 33K molecule containing IR-ACTH, IR-gamma 3MSH, and IR-beta END, presumed to be proOLMC, and a variety of 15-27K presumed biosynthetic intermediates were found in both normal pars distalis and pars intermedia. The pars intermedia adenoma causing Cushing's syndrome contained high IR-peptide concentrations. Several differences in precursors were noted, including the presence of three larger presumed precursors (38.5K, 47K, and 63K) that had both ACTH and beta END immunoreactivities and both deletions and additions of 15-27K intermediates. The Cushing's horse's plasma peptides reflected tumor concentrations; 4.5K ACTH was modestly elevated, but the concentrations of CLIP, alpha MSH, beta MSH, gamma LPH, and beta END were dramatically increased. About 20% of plasma IR-ACTH and 5% of IR-beta MSH and IR-beta END were found as high molecular weight forms. Normal processing of horse proOLMC appears to be similar to that in other species, but may be altered in pars intermedia tumors of horses with Cushing's disease, the plasma of which contains disproportionately increased concentrations of pars intermedia proOLMC peptides.