To study the role of cGMP-dependent protein kinase I (cGKI) for cardiac contractility, force of contraction (Fc) was studied in electrically driven heart muscle from wild-type (WT) mice and from conventional and conditional cGKI knockout mice. Both 8-Br-cGMP and 8-pCPT-cGMP reduced Fc in cardiac muscle from juvenile WT but not from juvenile cGKI-null mutants. Similarly, the cGMP analogues reduced Fcin forskolin-stimulated ventricular muscle from WT mice but not from cGKI-null mutants. In contrast, carbachol reduced Fcin both groups of animals. 8-Br-cGMP reduced Fcalso in heart muscle from adult WT mice but not from adult cardiomyocyte-specific cGKI-knockout mice. These results demonstrate that cGKI mediates the negative inotropic effect of cGMP in the myocardium of juvenile and adult mice.