Arteriolar myogenic vasoconstriction occurs when increased stretch or membrane tension leads to smooth muscle cell depolarization and opening of voltage-gated Ca2+ channels. To prevent positive feedback and excessive pressure-induced vasoconstriction, studies in cerebral artery smooth muscle have suggested that activation of large conductance, Ca2+-activated K+ channels (BKCa) provides an opposing hyperpolarizing influence reducing Ca2+ channel activity. We have hypothesized that this mechanism may not equally apply to all vascular beds. To establish the existence of such heterogeneity in vascular reactivity, studies were performed on rat vascular smooth muscle (VSM) cells from cremaster muscle arterioles and cerebral arteries. Whole cell K+ currents were determined at pipette [Ca2+] of 100 nM or 5 μM in the presence and absence of the BKCa inhibitor, iberiotoxin (IBTX; 0.1 μM). Similar outward current densities were observed for the two cell preparations at the lower pipette Ca2+ levels. At 5 μM Ca2+, cremaster VSM showed a significantly (P< 0.05) lower current density compared to cerebral VSM (34.5 ± 1.9 vs 45.5 ± 1.7 pA pF−1 at +70 mV). Studies with IBTX suggested that the differences in K+ conductance at 5 μM intracellular [Ca2+] were largely due to activity of BKCa. 17β-Oestradiol (1 μM), reported to potentiate BKCa current via the channel's β-subunit, caused a greater effect on whole cell K+ currents in cerebral vessel smooth muscle cells (SMCs) compared to those of cremaster muscle. In contrast, the α-subunit-selective BKCa opener, NS-1619 (20 μM), exerted a similar effect in both preparations. Spontaneously transient outward currents (STOCs) were more apparent (frequency and amplitude) and occurred at more negative membrane potentials in cerebral compared to cremaster SMCs. Also consistent with decreased STOC activity in cremaster SMCs was an absence of detectable Ca2+ sparks (0 of 76 cells) compared to that in cerebral SMCs (76 of 105 cells). Quantitative PCR showed decreased mRNA expression for the β1 subunit and a decrease in the β 1: α ratio in cremaster arterioles compared to cerebral vessels. Similarly, cremaster arterioles showed a decrease in total BKCa protein and the β 1: α-subunit ratio. The data support vascular heterogeneity with respect to the activity of BKCa in terms of both β-subunit regulation and interaction with SR-mediated Ca2+ signalling.