Aims: The sarcomeric titin springs are a major determinant of myocardial distensibility and passive stiffness. Titin stiffness can be lowered acutely by cAMP-dependent protein kinase (PKA) or cGMP-dependent protein kinase (PKG) mediated phosphorylation of the cardiac-specific N2-B unique sequence (N2-Bus). Using site-directed mutagenesis, serine residue S469 near the COOH-terminus of the human cardiac N2-Bus was identified as a PKG and PKA phosphorylation site. However, this site is not conserved among species. We therefore tested for PKG-mediated phosphorylation of the N2-Bus of dogs and rodents. Methods: Online prediction routines (NetphosK; Scansite/Motifscan) were used to identify putative PKG-dependent phosphorylation sites in the dog, mouse, and rat N2-Bus. PKG-mediated phosphorylation of predicted sites was measured on recombinantly expressed N2-Bus from dog and mouse, as well as NH2-terminal and COOH-terminal fragments, incubated with cGMP-activated PKG-Ib. Results: Prediction algorithms suggested the presence of PKG-dependent phosphorylation sites in dog, mouse, and rat N2-Bus. Phospho-specific protein stain (ProQ-diamond) showed phosphorylation of both dog and mouse N2-Bus, which was confirmed by autoradiography. PKG phosphorylated an NH2-terminal fragment of the dog N2-Bus and a COOH-terminal fragment of the mouse N2-Bus. Conclusion: The N2-Bus can be phosphorylated by PKG in humans and various other mammalian species. As PKG-mediated phosphorylation of the N2-Bus lowers cardiac titin stiffness in human myocardium, a similar mechanism may be generally active in mammalian hearts. We suggest that PKG-mediated phosphorylation of the titin N2-Bus is a conserved regulatory event to acutely adjust passive myocardial stiffness.