Vascular smooth muscle contractile state is regulated by intracellular calcium levels. Nitric oxide causes vascular relaxation by stimulating production of cyclic GMP, which activates type I cGMP-dependent protein kinase (PKGI) in vascular smooth muscle cells (VSMC), inhibiting agonist-induced intracellular Ca2+ mobilization ([Ca2+]in). The relative roles of the two PKGI isozymes, PKGI and PKGI, in cyclic GMP-mediated inhibition of [Ca2+]in in VSMCs are unclear. Here we investigate the ability of PKGI isoforms to inhibit [Ca2+]in in response to VSMC activation. Stable CHO cell lines expressing PKGI or PKGI were created and the ability of PKGI isoforms to inhibit [Ca2+]in in response to thrombin receptor stimulation was examined. In CHO cells stably expressing PKGI or PKGI, 8-Br-cGMP activation suppressed [Ca2+]in by thrombin receptor activation peptide (TRAP) by 98 +/- 1% vs. 42 +/- 5%, respectively (p < 0.002). Immunoblotting studies of cultured human VSMC cells from multiple sites using PKGI- and PKGI-specific antibodies showed PKGI is the predominant VSMC PKGI isoform. [Ca2+]in following thrombin receptor stimulation was examined in the absence or presence of cyclic GMP in human coronary VSMCs cells (Co403). 8-Br-cGMP significantly inhibited TRAP-induced [Ca2+]in in Co403, causing a four-fold increase in the EC50 for [Ca2+]in. In the absence of 8-Br-cGMP, suppression of PKGI levels by RNAi led to a significantly greater TRAP-stimulated rise in [Ca2+]in as compared to control RNAi-treated Co403 cells. In the presence of 8-Br-cGMP, the suppression of PKGI expression by RNAi led to the complete loss of cGMP-mediated inhibition of [Ca2+]in. Adenoviral overexpression of PKGI in Co403 cells was unable to alter TRAP-stimulated Ca2+ mobilization either before or after suppression of PKGI expression by RNAi. These results support that PKGI is the principal cGMP-dependent protein kinase isoform mediating inhibition of VSMC activation by the nitric oxide/cyclic GMP pathway.