8-Chloro-cyclic AMP (8-Cl-cAMP), which is known to induce growth inhibition, apoptosis, and differentiation in various cancer cell lines, has been studied as a putative anticancer drug. However, the mechanism of anticancer activities of 8-Cl-cAMP has not been fully understood. Previously, we reported that the 8-Cl-cAMP-induced growth inhibition is mediated by protein kinase C (PKC) activation. In this study, we found that p38 mitogen-activated protein kinase (MAPK) also plays important roles during the 8-Cl-cAMP-induced growth inhibition and apoptosis. SB203580 (a p38-specific inhibitor) recovered the 8-Cl-cAMP-induced growth inhibition and apoptosis, whereas other MAPK inhibitors, such as PD98059 (an extracellular signal-regulated kinase-specific inhibitor) and SP600125 (a c-Jun NH2-terminal kinase-specific inhibitor), had no effect. The phosphorylation (activation) of p38 MAPK was increased in a time-dependent manner after 8-C1-cAMP treatment. Furthermore, SB203580 was able to block PKC activation induced by 8-Cl-cAMP. However, PKC inhibitor (GF109203x) could not attenuate p38 activation, indicating that p38 MAPK activation is upstream of PKC activation during the 8-Cl-cAMP-induced growth inhibition. 8-Chloroadenosine, a metabolite of 8-Cl-cAMP, also activated p38 MAPK and this activation was blocked by adenosine kinase inhibitor. These results suggest that 8-Cl-cAMP exerts its anticancer activity through p38 MAPK activation and the metabolite(s) of 8-Cl-cAMP mediates this process.