Protein kinase G-I (PKG-I) activation is essential for vascular relaxation; however, its quantitative analysis in intact cells has been difficult. To overcome this difficulty, a monoclonal antibody, VASP-16C2, was recently developed that detects phosphorylated serine residue 239 of vasodilator-stimulated phosphoprotein (VASP), a substrate of PKG-I. In this study, we used this antibody to examine (i) possible functional differences between the alpha and beta isoforms of PKG-I, (ii) ability of cAMP to activate PKG-I, as compared to cGMP, the principal PKG-I-activating cyclic nucleotide, and (iii) time course and levels of PKG-I activation in vascular smooth muscle cells (VSMC) of young and old rats. We created COS-7 cell clones that overexpressed PKG-I alpha or PKG-I beta, treated them with cAMP or cGMP, and analyzed their cell lysates for reactivity with VASP-16C2. The results showed that PKG-Ia phosphorylated VASP at a higher level than PKG-I beta, and cAMP was slightly weaker than cGMP in PKG-I activation. VSMC of young rats responded to cAMP or cGMP stimulation in a dose-dependent manner with increasing levels of PKG-I activation. The response was detected within 10 min and continued for at least 24 h. In contrast, VSMC of old rats showed no PKG-I activation during the first hour of cAMP or cGMP stimulation and, at 24 h these cells showed only low-level PKG-I activation. We propose that the reduced PKG-I activation may explain why vascular relaxation is decreased in older individuals.