AMP-activated protein kinase (AMPK) has been identified as a regulator of gene transcription, increasing mitochondrial proteins of oxidative metabolism as well as hexokinase expression in skeletal muscle. In mice, muscle specific knockout of LKB1, a component of the upstream kinase of AMPK, prevents contraction- and AICAR-induced activation of AMPK in skeletal muscle, and the increase in hexokinase II protein which is normally observed with chronic AICAR activation of AMPK. Since previous reports show a cyclic AMP response element in the promoter region of the hexokinase II gene, we hypothesized that the cyclic AMP response element binding protein (CREB) family of transcription factors could be targets of AMPK. Using radioisotopic kinase assays, we found that recombinant and rat liver and muscle AMPK phosphorylated CREB1 at the same site as cAMP-dependent protein kinase (PKA). AMPK was also found to phosphorylate ATF1, CREM, and CREBL2, but not ATF2. Treatment of HEK293 cells stably transfected with a CREB-driven luciferase reporter with AICAR increased luciferase activity approximately three fold over a 24 hour time course. This increase was blocked with compound C, an AMPK inhibitor. In addition, AICAR-induced activation of AMPK in incubated rat epitrochlearis muscles resulted in an increase in both phospho-ACC and phospho-CREB. We conclude that CREB and related proteins are direct downstream targets for AMPK and are therefore likely involved in mediating some effects of AMPK on expression of genes having a cyclic AMP responsive element in their promoters.