In the present study we investigated whether norepinephrine, which stimulates melatonin biosynthesis in the mammalian pineal organ, causes phosphorylation of the cyclic AMP responsive element binding protein (CREB) in rat pinealocytes. Cells isolated from the pineal organ of adult male rats and cultured on coated coverslips were treated with norepinephrine, beta- or alpha 1-adrenergic agonists for 12, 5, 10, 20, 30, 60 or 300 min and then immunocytochemically analyzed with an antibody against phosphorylated CREB (p-CREB). Treatment with norepinephrine or beta-adrenergic agonists resulted in a similar, time-dependent induction of p-CREB immunoreactivity, exclusively found in cell nuclei. The alpha 1-adrenergic agonist phenylephrine did not induce p-CREB immunoreactivity at low doses (0.1 microM) or when high doses (10 microM) were applied in combination with a beta-antagonist (propranolol, 0.1 microM). This indicates that induction of CREB phosphorylation is elicited by beta-adrenergic receptor stimulation. The response was first seen after 10 min and reached a maximum after 30 to 60 min when more than 90% of the cells displayed p-CREB immunoreactivity. The intensity of the p-CREB immunoreactivity showed marked cell-to-cell variation, but nearly all immunoreactive cells were identified as pinealocytes by double-labeling with an antibody against the S-antigen, a pinealocyte-specific marker. The results show that norepinephrine stimulation induces p-CREB immunoreactivity by acting upon beta-adrenergic receptors in virtually all rat pinealocytes. The findings support the notion that phosphorylation of CREB is a rather rapid and uniform response of pinealocytes to noradrenergic stimulation and thus is an important link between adrenoreceptor activation and subsequent gene expression in the rat pineal organ.