The synthesis and secretion of various intermediate pituitary proteins was studied by using dispersed intermediate pituitary cell suspensions. Control studies indicated that the isolated cells were obtained in good yield and that after more than 24 h in culture the isolated cells continued to synthesize a collection of proteins similar to those found in freshly extracted intermediate pituitary tissue. Rat intermediate pituitary cells synthesized a molecule (Mr = 30,000; called 30K) that contained antigenic determinants for beta-endorphin, gamma-lipotropin, corticotropin (ACTH), and 16K fragment (the NH2-terminal region of mouse tumor cell pro-ACTH/endorphin). This 30K molecule, two high molecular weight forms of ACTH(13K and 20K), and 16K fragment were all shown to be glycoproteins. Continuous labeling and pulse-chase incubations were used to define the intracellular biosynthetic processing of the 30K molecule. After a 15-min pulse incubation the 30K molecule was the only labeled protein containing antigenic determinants for beta-endorphin, gamma-lipotropin, ACTH, or 16K fragment. A beta-lipotropin-like molecule served as a biosynthetic intermediate in the production of proteins similar to beta-endorphin and gamma-lipotropin. Methionine-enkephalin and alpha-endorphin were not major products in the intermediate lobe cells. Molecules similar to alpha-melanocyte-stimulating hormone and corticotropin-like intermediate lobe peptide (ACTH(18-39)) were also derived from the same 30K molecule; 20K ACTH served as a biosynthetic intermediate in this conversion. In rat intermediate pituitary cells ACTH(1-39) was not a major final product of the intracellular biosynthetic processing of the 30K molecule. The 30K molecule also served as a precursor to a protein similar to mouse tumor cell 16K fragment and related smaller proteins. With rat intermediate pituitary cells, pulse-chase experiments utilizing [35S]methionine demonstrated almost quantitative conversion of the 30K precursor into labeled proteins similar to beta-endorphin and alpha-melanocyte-stimulating hormone. In the absence of added secretagogues, small amounts of all of the smaller proteins derived from the 30K precursor were secreted coordinately into the culture medium.