A growing body of evidence has demonstrated that p38 mitogen-activated protein kinase (MAPK) has a crucial role in various physiological and pathological processes mediated by beta(2)-adrenergic receptors (beta(2)-ARs). However, the detailed mechanism of beta(2)-ARs-induced p38 MAPK activation has not yet been fully defined. The present study demonstrates a novel kinetic model of p38 MAPK activation induced by beta(2)-ARs in human embryonic kidney 293A cells. The beta(2)-AR agonist isoproterenol induced a time-dependent biphasic phosphorylation of p38 MAPK: the early phase peaked at 10 min, and was followed by a delayed phase that appeared at 90 min and was sustained for 6 h. Interestingly, inhibition of the cAMP/protein kinase A (PKA) pathway failed to affect the early phosphorylation but abolished the delayed activation. By contrast, silencing of beta-arrestin-1 expression by small interfering RNA inhibited the early phase activation of p38 MAPK. Furthermore, the NADPH oxidase complex is a downstream target of beta-arrestin-1, as evidenced by the fact that isoproterenol-induced Rac1 activation was also suppressed by beta-arrestin-1 knockdown. In addition, early phase activation of p38 MAPK was prevented by inactivation of Rac1 and NADPH oxidase by pharmacological inhibitors, overexpression of a dominant negative mutant of Rac1, and p47(phox) knockdown by RNA interference. Of note, we demonstrated that only early activation of p38 MAPK is involved in isoproterenol-induced F-actin rearrangement. Collectively, these data suggest that the classic cAMP/PKA pathway is responsible for the delayed activation, whereas a beta-arrestin-1/Rac1/NADPH oxidase-dependent signaling is a heretofore unrecognized mechanism for beta(2)-AR-mediated early activation of p38 MAPK.