Equine Cushing's disease is caused by an adenomatous hyperplasia of the intermediate pituitary which secretes high levels of beta-endorphin, ACTH, and other peptide derivatives of POMC. In the present study we found that plasma and cerebrospinal fluid immunoreactive beta-endorphin (i beta-endorphin) levels were 60- and 120-fold higher than control values in horses with Cushing's disease. There were no significant differences in intermediate lobe i beta-endorphin concentrations, although anterior lobe i beta-endorphin was significantly reduced in Cushing's horses, presumably because high levels of circulating glucocorticoids inhibit POMC biosynthesis in corticotrophs. Although the i beta-endorphin concentration of the tumors was not different from that in normal tissue, the posttranslational processing of beta-endorphin in the two tissues differed significantly. In controls, beta-endorphin-(1-31) was extensively processed to N-acetyl-beta-endorphin-(1-31), -(1-27), and -(1-26) and des-acetyl beta-endorphin-(1-27). N-Acetyl-beta-endorphin-(1-27) was the predominant form, constituting 57% of the total i beta-endorphin, whereas beta-endorphin-(1-31) was quantitatively minor (less than 7% of the total immunoreactivity. In adenomatous pituitaries, the processing of beta-endorphin was restricted, significantly increasing the proportions of beta-endorphin-(1-31) and N-acetyl-beta-endorphin-(1-31) and lowering the amounts of N-acetyl-beta-endorphin-(1-27) and -(1-26). These changes in peptide processing were associated with markedly reduced levels of dopamine, suggesting that the dopaminergic neurons that normally control intermediate lobe secretion no longer innervate the hyperplastic tissue. These findings are consistent with evidence that the dopaminergic innervation of the intermediate pituitary regulates the posttranslational processing and release of beta-endorphin.