In the present study, we tested the hypothesis that the activation of imidazoline I₁-receptor, which is coupled to phosphatidylcholine-specific phospholipase C, results in downstream activation of mitogen-activated protein kinase (p42^mapk and p44^mapk isoforms) in PC12 cells. PC12 cells pretreated with nerve growth factor (50 ng/ml, 48 h) to initiate neuronal differentiation were incubated with [methyl-³H]choline and [³H]myristate. Activation of imidazoline I₁ receptor by rilmenidine (10 μM) caused time-dependent increases in diacylglycerol accumulation and phosphocholine release. The Western blotting analysis showed that rilmenidine (10 μM) produced a time-dependent activation of p42^mapk and p44^mapk that reached its maximum at 15 min and returned to control levels after 30 min. This finding was confirmed by immunofluorescence labeling of activated mitogen-activated protein kinase in the same model system. Efaroxan (imidazoline I₁-receptor antagonist) or tricyclodecan-9-yl-xanthogenate (D609, phosphatidylcholine-specific phospholipase C inhibitor) attenuated the phosphorylation of p42^mapk and p44^mapk induced by rilmenidine. Nerve growth factor-induced phosphorylation of both mitogen-activated protein kinase isoforms was not affected by D609. These results support the hypothesis that the activation of the imidazoline I₁ receptor coupled phosphatidylcholine-specific phospholipase C results in the downstream activation of mitogen-activated protein kinase.