d-Amphetamine and methylphenidate are widely used in the treatment of attention-deficit/hyperactivity disorder. Both drugs increase extracellular norepinephrine and dopamine in the prefrontal cortex, where they are believed to exert their therapeutic effects. However, the molecular mechanisms underlying their action are poorly understood. To investigate the intracellular signaling pathways activated by d-amphetamine and methylphenidate in the prefrontal cortex in vivo in mice, we measured the cAMP-dependent Ser845 phosphorylation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor GluR1 subunit and the active form of extracellular signal-regulated kinase (ERK). Administration of d-amphetamine (5-10 mg/kg) or methylphenidate (10-20 mg/kg) increased phosphorylation of GluR1. Basal and d-amphetamine-induced GluR1 phosphorylation was reduced by propranolol, a general beta-adrenoceptor antagonist, and betaxolol, a beta1-antagonist, but not by (+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol (ICI-118,515), a beta2-antagonist. The effect of methylphenidate was also blocked by propranolol and betaxolol. The d-amphetamine effect was slightly potentiated by prazosin, an alpha1-adrenoceptor antagonist, and mimicked by yohimbine, an alpha2 antagonist. Blockade of dopamine or N-methyl-d-aspartate (NMDA) receptors or serotonin depletion had no effect on d-amphetamine-induced GluR1 phosphorylation. d-amphetamine but not methylphenidate increased ERK phosphorylation. This effect required multiple signaling pathways because it was blocked by beta1- and alpha1-adrenoceptor antagonists, by dizocilpine maleate (MK801), an NMDA antagonist, and by serotonin depletion. In contrast, blockade of dopamine receptors had no effect on d-amphetamine-induced ERK phosphorylation. Propranolol and betaxolol increased the hyperlocomotion produced by d-amphetamine and methylphenidate. Thus, both d-amphetamine and methylphenidate potently activate the cAMP pathway in the prefrontal cortex through beta1-adrenergic receptors. This activation could have behavioral consequences and contribute to the treatment of attention-deficit/hyperactivity disorder.