Protein IIIa (Mr approximately 74,000) and protein IIIb (Mr approximately 55,000), referred to collectively as protein III, are synaptic vesicle-associated phosphoproteins found in all regions of the rat nervous system and in the rat adrenal medulla. In the present study, the presence of protein III and the regulation of its phosphorylation were examined in chromaffin cells isolated from bovine adrenal medullae. Protein III was present in chromaffin cells isolated and purified from bovine adrenal medullae. The levels of protein III were moderately enriched in purified chromaffin cells compared with whole adrenal medullae. Preincubation of chromaffin cells with 32PO4 led to the endogenous phosphorylation of protein III, and phosphopeptide maps of chromaffin cell protein III were similar to those of protein III from bovine cerebral cortex. Treatment of the chromaffin cells with ACh produced calcium-dependent increases in both the phosphorylation of protein III and the release of 3H-norepinephrine. These effects of ACh were mimicked by nicotine but not by muscarine. Other secretagogues (elevated K+, veratridine, Ba2+) also increased both the phosphorylation of protein III and the release of 3H-norepinephrine. However, detailed characterization of the secretagogue-induced increases in protein III phosphorylation and 3H-norepinephrine secretion suggested that protein III phosphorylation was more directly associated with an increase in intracellular calcium than with secretion per se.