Nitric oxide (NO) donors regulate K-Cl cotransport (KCC) activity and cotransporter-1 and -3 (KCC1 and KCC3) mRNA expression in sheep erythrocytes and in primary cultures of rat vascular smooth muscle cells (VSMCs), respectively. Here, we used NONOates as rapid and slow NO releasers, to provide direct evidence implicating NO as a regulator of KCC3 gene expression at the mRNA level. In addition, we used the expression of KCC3 mRNA to further investigate the mechanism of action of these NO donors at the cellular level. Treatment of VSMCs with rapid NO releasers, like NOC-5 and NOC-9, as well as with the direct NO-independent soluble guanylyl cyclase (sGC) stimulator YC-1, acutely increased KCC3 mRNA expression in a concentration- and time-dependent manner. The slow NO releaser NOC-18 had no effect on KCC3 gene expression. A specific NO scavenger completely prevented the NONOate-induced KCC3 mRNA expression. Inhibition of sGC with LY83583 blocked the NONOate-, and YC-1-induced KCC3 mRNA expression. This study shows that in primary cultures of rat VSMCs fast NO releasers, NOC-9 and NOC-5 but not the slow NO releaser, NOC-18 acutely up-regulate KCC3 mRNA expression in a NO/sGC-dependent manner.