The c-Jun NH2-terminal kinases (JNKs) are important stress-responsive kinases. They regulate cellular activities by sequential phosphorylation and activation through a mitogen-activated protein kinase cascade, whereas JNKs activation is altered in response to various stressors. In the present study, we used immunoblotting to assess the effect of 21 day of social isolation as the chronic stressor, either sole and in combination with 2 h of acute immobilization or cold (4°C) stress on circulating corticosterone level and phosphorylation status of p46 (phospho-p46/total p46) and p54 (phospho-p54/total p54) JNK isoforms in the cytosolic and nuclear fraction of the prefrontal cortex and hippocampus of male Wistar rats. Also, the phosphorylation status of JNK nuclear down-stream target c-Jun (p-c-Jun/c-Jun) on Ser63 was examined. Both acute stressors with elevated CORT levels led to increased phosphorylation status of cytosolic p54 JNK isoforms but not p46 JNK isoforms only in the hippocampus and no change in phosphorylation status of c-jun in both brain regions. Chronic isolation with unaltered CORT level and reduced responsiveness to novel acute stressors, led to unchanged or reduced phosphorylation status of p46 and p54 JNK isoforms in both fractions and both brain regions, whereas the decrease of c-Jun phosphorylation status was found only in the prefrontal cortex. Our results suggest that compromised JNKs activation following chronic isolation may lead to interruption of JNK signaling, which could be related with neuropsychiatric disorders such as depression or long-lasting neuronal remodeling.