Titin is a major determinant of myocardial elasticity and passive stiffness. Apart from variations in the titin-isoform composition, 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). A 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, thus questioning the physiological relevance of titin phosphorylation in commonly used animal models of heart failure. Expression of cardiac PKG was confirmed in tissue samples from human, rat, mouse, and dog hearts. Using online prediction routines we identified putative PKG-targeted phosphorylation sites in dog and mouse/rat titin N2-Bus. PKG-mediated phosphorylation of the predicted sites was tested by incubation of recombinantly expressed N2-Bus, and NH2-terminal and COOH-terminal peptides of dog and mouse N2-Bus with cGMP-activated PKG-Ib. A phospho-specific protein stain showed phosphorylation of titin N2-Bus in peptides from both investigated species. Phosphorylation of the dog N2-Bus peptides was further confirmed by autoradiography using radioactively labeled ATP for the kinase assay. We found that PKG phosphorylated an NH2-terminal fragment of the dog N2-Bus, and a COOH-terminal fragment of the mouse/rat sequence. We presume that phosphorylation of a single site in the N2-Bus is likely to cause a similar reduction in titin stiffness as observed for the human titin sequence, regardless to the exact site of phosphorylation. In summary, our results suggest that PKG-mediated phosphorylation of titin N2-Bus is an important regulatory event not only in human but in all mammalian hearts.