Nitrate tolerance (NT) in hypertension is attributed to reduced activity of soluble guanylyl cyclase (sGC). We examined NT in basilar artery vascular smooth muscle cells (VSMCs) from control rats, rats infused with angiotensin II (Ang; 240 μg/kg per hour for 4 days), which were normotensive, and Ang-hypertensive rats (AHR; 240 μg/kg per hour for 28 days). Ca²⁺-activated K⁺ (Maxi-K) channels in VSMCs from AHR showed reduced activation by NO donor, consistent with NT. The concentration-response relationship for 8-Br-cGMP was shifted 2.5-fold to the right, indicating that abnormal sGC alone could not account for NT. Inside-out patches from AHR showed normal activation with exogenous cGMP-dependent protein kinase I (cGKI), suggesting no abnormality downstream of cGKI. We hypothesized that the reduction in apparent affinity of 8-Br-cGMP for cGKI in AHR might be due to a change in relative amounts of cGKIα versus cGKIβ, since cGKIβ is less sensitive to cGMP activators than cGKIα. This was substantiated by showing the following in AHR: (1) reduced effect of the cGKIα-selective activator 8-APT-cGMP; (2) reduced total cGKI protein (both isoforms), but an increase in cGKIβ protein in quantitative immunofluorescence and Western blots; (3) similar changes in cGKI isoforms immunoisolated with Maxi-K channels; and (4) a large increase in cGKIβ mRNA and a decrease in cGKIα mRNA in real-time PCR and Northern blots. Upregulation of cytosolic cGKIβ was evident 4 days after Ang infusion, before development of hypertension. Our data identify a functional role for cGKIβ in VSMCs previously ascribed exclusively to cGKIα. Ang-induced alternative splicing of cGKI represents a novel mechanism for reducing sensitivity to NO/cGMP.