Type I cyclic guanosine 3′,5′-monophosphate (cGMP)-dependent protein kinase (PKG) is involved in the nitric oxide/cGMP signaling pathway. PKG has been identified in many different species, ranging from unicelõlular organisms to mammals. The enzyme serves as one of the major receptor proteins for intracellular cGMP and controls a variety of cellular responses, ranging from smooth-muscle relaxation to neuronal synaptic plasticity. In the absence of a crystal structure, the three-dimensional structure of the homodimeric 152-kDa kinase PKG is unknown; however, there is evidence that the kinase adopts a distinct cGMP-dependent active conformation when compared to the inactive conformation. We performed mass-spectrometry-based hydrogen/deuterium exchange experiments to obtain detailed information on the structural changes in PKG Iα induced by cGMP activation. Site-specific exchange measurements confirmed that the autoinhibitory domain and the hinge region become more solvent exposed, whereas the cGMP-binding domains become more protected in holo-PKG (dimeric PKG saturated with four cGMP molecules bound). More surprisingly, our data revealed a specific disclosure of the substrate-binding region of holo-PKG, shedding new light into the kinase-activation process of PKG.