SNARE proteins, syntaxin-1A (Syn-1A) and SNAP-25, inhibit delayed rectifier K⁺ channels, Kv1.1 and Kv2.1 in secretory cells. We showed previously that the mutant open conformation of Syn-1A (Syn-1A L165A/E166A) inhibits Kv2.1 channels more optimally than wild type Syn-1A. In this report we examined whether Syn-1A in its wild type and open conformations would exhibit similar differential actions on the gating of Kv1.2, a major delayed rectifier K⁺ channel in non-secretory smooth muscle cells and some neuronal tissues. In co-expression and acute dialysis studies, wild type Syn-1A inhibited Kv1.2 current magnitude. Of interest, wild type Syn-1A caused a right-shift in the activation curves of Kv1.2 without affecting its steady-state availability, an inhibition profile opposite to its effects on Kv2.1 (steady-state availability reduction without changes in voltage-dependence of activation). Also, although both wild type and open form Syn-1A bound equally well to Kv1.2 in an expression system, open form Syn-1A failed to reduce Kv1.2 current magnitude or affect its gating. This is in contrast to the reported more potent effect of open form Syn-1A on Kv2.1 channels in secretory cells. This finding together with the absence of Munc18 and/or 13-1 in smooth muscles suggested that a change to an open conformation Syn-1A, normally facilitated by Munc18/13-1, is not required in non-secretory smooth muscle cells. Taken together with previous reports, our results demonstrate the multiplicity of gating inhibition of different Kv channels by Syn-1A, and is compatible with versatility of Syn-1A modulation of repolarization in various secretory and non-secretory (smooth muscle) cell types.