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In vivo protein kinases A and G (PKA and PKG) coordinately phosphorylate a broad range of substrates to mediate their various physiological effects. The functions of many of these substrates have yet to be defined genetically. Herein we show a role for smoothelin-like protein 1 (SMTNL1), a novel in vivo target of PKG/PKA, in mediating vascular adaptations to exercise. Aortas from smtnl1 ؊/؊ mice exhibited strikingly enhanced vasorelaxation before exercise, similar in extent to that achieved after endurance training of wild-type littermates. Additionally, contractile responses to ␣-adrenergic agonists were greatly attenuated. Immunological studies showed SMTNL1 is expressed in smooth muscle and type 2a striated muscle fibers. Consistent with a role in adaptations to exercise, smtnl1 ؊/؊ mice also exhibited increased type 2a fibers before training and better performance after forced endurance training compared smtnl1 ؉/؉ mice. Furthermore, exercise was found to reduce expression of SMTNL1, particularly in female mice. In both muscle types, SMTNL1 is phosphorylated at Ser-301 in response to adrenergic signals. In vitro SMTNL1 suppresses myosin phosphatase activity through a substrate-directed effect, which is relieved by Ser-301 phosphorylation. Our findings suggest roles for SMTNL1 in cGMP/cAMP-mediated adaptations to exercise through mechanisms involving direct modulation of contractile activity.The contractile state of smooth muscle (SM) 2 is largely governed by phosphorylation of myosin regulatory light chain (LC20), which in turn is regulated by the opposing activities of myosin light chain kinase (MLCK) and myosin phosphatase, SMPP1M (1). In SM, both MLCK and SMPP1M are regulated, thereby enabling homeostatic control of contractile activity (2). Exquisite control is necessary because of the essential roles of SM in many physiological processes, such as maintenance of blood pressure. In most SMs, release of cyclic nucleotides (cGMP/cAMP), in response to hormonal or neuronal stimulation, promote relaxation either by lowering intracellular [Ca 2ϩ ] or desensitizing the muscle to Ca 2ϩ by inhibiting MLCK and/or activating SMPP1M (2).Targeted deletions of both PKA and PKG in mice produce profound phenotypes, underscoring the importance of these kinases in many physiological processes (3, 4). In vivo, both kinases are known to selectively target a discrete number of substrates and current thinking suggests that selective targeting is the means by which these broadly acting enzymes bring about coordinated physiological responses (5). A few groups have begun to test this hypothesis by selectively deleting PKA/PKG targets in mice. Schlossmann et al. (6) demonstrated a major role for IRAG (inositol 1,4,5-trisphosphate receptor 1 IP3R1-associated protein, with exon 12 deleted by removing the 1,4,5-trisphosphate receptor binding domain) in PKG-mediated regulation of [Ca 2ϩ ] in SM. Disruption of IRAG resulted in a selective loss of signaling response. Other PKG/PKA-mediated responses were largely intact, contrasting wi...