From its position in presynaptic nerve terminals, the large conductance Ca²⁺-activated K⁺ channel, Slo, regulates neurotransmitter release. Several other ion channels known to control neurotransmitter release have been implicated in physical interactions with the neurotransmitter release machinery. For example, the Ca_v2.2 (N-type) Ca²⁺ channel binds to and is modulated by syntaxin-1A and SNAP-25. Furthermore, a close juxtaposition of Slo and Ca_v2.2 is presumed to be necessary for functional coupling between the two channels, which has been demonstrated in neurons. We report that Slo exhibits a strong association with syntaxin-1A. Robust coimmunoprecipitation of Slo and syntaxin-1A occurs from transfected HEK293 cells as well as from brain. However, despite this strong interaction and the known association between syntaxin-1A and the II-III loop of Ca_v2.2, these three proteins do not coimmunoprecipitate in a trimeric complex from transfected HEK293 cells. The Slo-syntaxin-1A coimmunoprecipitation is not significantly influenced by [Ca²⁺]. Multiple relatively weak interactions may sum up to a tight physical coupling of full-length Slo with syntaxin-1A: the C-terminal tail and the S0-S1 loop of Slo each coimmunoprecipitate with syntaxin-1A. The presence of syntaxin-1A leads to reduced Slo channel activity, due to an increased V_1/2 for activation in 100 nM, 1 µM and 10 µM Ca²⁺, reduced voltage-sensitivity in 1 µM Ca²⁺ and slower rates of activation in 10 µM Ca²⁺. Potential physiological consequences of the interaction between Slo and syntaxin-1A include enhanced excitability, through modulation of Slo channel activity, and reduced neurotransmitter release, due to disruption of syntaxin-1A's binding to the Ca_v2.2 II-III loop.