The effects of p38 mitogen-activated protein kinase (p38MAPK) inhibitors on the adrenergic-stimulated cyclic nucleotide production in rat pinealocytes were investigated. Treatment with SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)IH-imidazole] and SB203580 [4-(4-fluoropheny)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)IH-imidazole] (1-100 microM), two pyridinyl imidazole compounds that inhibit p38MAPK, as well as SB202474 [4-(ethyl)-2-(4-methoxyphenyl)-5-(4-pyridyl)IH-imidazole], an inactive analog, was effective in potentiating norepinephrine- and isoproterenol-stimulated cyclic AMP (cAMP) and cyclic GMP (cGMP) accumulation in a concentration-dependent manner. All three compounds caused a greater increase in the cGMP than the cAMP response, with SB202474 being substantially more potent than the two active analogs. At 100 microM, SB202474 potentiated the isoproterenol-stimulated cAMP and cGMP accumulation by 65 and 500%, respectively. Pharmacological studies indicated that the potentiating effect of SB202474 was independent of protein kinase C activation, intracellular calcium elevation, or serine/threonine phosphatase inhibition, three pathways known to potentiate the beta-adrenergic-stimulated cyclic nucleotide responses in rat pinealocytes. In contrast, the potentiating effect of SB202474 was abolished in the presence of a phosphodiesterase inhibitor, isobutylmethylxanthine. At 100 microM, all three compounds inhibited cAMP- and cGMP-phosphodiesterase activities by 50 and 80%, respectively. These results suggest that the commonly used p38MAPK inhibitors can modulate cyclic nucleotide responses through phosphodiesterase inhibition, a mechanism that appears to be independent of p38MAPK inhibition.