Regulation of cAMP-dependent protein phosphorylation by acute and chronic morphine was studied in the rat locus coeruleus (LC), a brain region that appears to play an important role in mediating morphine action in animals, including humans. Rats were treated chronically with morphine under conditions known to induce states of tolerance and dependence. Protein phosphorylation was then studied in extracts of LC and other brain regions with back-phosphorylation assays under different conditions and with 1- and 2-dimensional electrophoretic procedures. Evidence was obtained to suggest that chronic morphine increased the phosphorylation state of proteins of 165, 55, and 14-20 kDa and increased the total amount of proteins of 145, 71, 62, 58, and 51 kDa in the LC. Concomitant treatment of rats with naltrexone, an opiate receptor antagonist, blocked the ability of morphine to influence each of these proteins, indicating that morphine regulation of protein phosphorylation occurred through the specific activation of opiate receptors. Regulation of 165, 71, 62, 58, 55, and 51 kDa by chronic morphine was specific to the LC among the brain regions studied, whereas regulation of 145 and 14-20 kDa was also observed in the frontal cortex, neostriatum, and dorsal raphe. Most of the phosphoproteins whose phosphorylation state or total amount was increased by chronic morphine treatment in the LC in vivo were also shown to have their phosphorylation state decreased by acute morphine treatment in isolated LC nuclei ex vivo. In addition, the phosphorylation state of most of these morphine-regulated phosphoproteins was stimulated by forskolin or cAMP analogs in isolated LC and by cAMP in broken cell preparations of this brain region, supporting the view that these proteins are physiological substrates for cAMP-dependent protein kinase in the LC. Phosphoproteins regulated by morphine and cAMP were designated "MARPPs," morphine- and cAMP-regulated phosphoproteins, whereas those regulated by morphine but not by cAMP were designated "MRPPs." Taken together, the results of this study indicate that chronic morphine produces specific alterations in cAMP-dependent protein phosphorylation in the LC and raise the possibility that regulation of these specific phosphoproteins contributes to the development of tolerance and/or dependence in these neurons.