Abstract β1-Subunits enhance the gating properties of large-conductance Ca(2+)-activated K(+) channels (BKCa) formed by α-subunits. In arterial vascular smooth muscle cells (VSMCs), β1-subunits are vital in coupling SR-generated Ca(2+) sparks to BKCa activation, affecting contractility and blood pressure. Studies in cremaster and cerebral VSMCs show heterogeneity of BKCa activity due to apparent differences in the functional β1-subunit:α-subunit ratio. To define these differences, studies were conducted at the single-channel level while siRNA was used to manipulate specific subunit expression. β1 modulation of the α-subunit Ca(2+) sensitivity was studied using patch-clamp techniques. BKCa channel normalized open probability (NPo) versus membrane potential (Vm) curves were more left-shifted in cerebral versus cremaster VSMCs as cytoplasmic Ca(2+) was raised from 0.5 to 100 μm. Calculated V1/2 values of channel activation decreased from 72.0 ± 6.1 at 0.5 μm Ca(2+)i to -89 ± 9 mV at 100 μm Ca(2+)i in cerebral compared with 101 ± 10 to -63 ± 7 mV in cremaster VSMCs. Cremaster BKCa channels thus demonstrated an ∼2.5-fold weaker apparent Ca(2+) sensitivity such that at a value of Vm of -30 mV, a mean value of [Ca(2+)]i of 39 μm was required to open half of the channels in cremaster versus 16 μm [Ca(2+)]i in cerebral VSMCs. Further, shortened mean open and longer mean closed times were evident in BKCa channel events from cremaster VSMCs at either -30 or 30 mV at any given [Ca(2+)]. β1-Subunit-directed siRNA decreased both the apparent Ca(2+) sensitivity of BKCa in cerebral VSMCs and the appearance of spontaneous transient outward currents. The data are consistent with a higher ratio of β1-subunit:α-subunit of BKCa channels in cerebral compared with cremaster VSMCs. Functionally, this leads both to higher Ca(2+) sensitivity and NPo for BKCa channels in the cerebral vasculature relative to that of skeletal muscle.