The large conductance Ca(2+)-activated K(+) (BK(Ca)) channel in vascular smooth muscle cell (VSMC) is an important potassium channel that can regulate vascular tone. Recent work has demonstrated that abnormalities in BK(Ca) channel function are associated with changes in cell proliferation and the onset of vascular disease. However, until today there are rare reports to show whether this channel is involved in VSMC proliferation in response to fluid shear stress (SS). Here we investigated a possible role of BK(Ca) channel in VSMC proliferation under laminar SS. Rat aortic VSMCs were plated in parallel-plate flow chambers and exposed to laminar SS with varied durations and magnitudes. VSMC proliferation was assessed by measuring proliferating cell nuclear antigen (PCNA) expression and DNA synthesis. BK(Ca) protein and gene expression was determined by flow cytometery and RT-PCR. The involvement of BK(Ca) in SS-induced inhibition of proliferation was examined by BK(Ca) inhibition using a BK(Ca) specific blocker, iberiotoxin (IBTX), and by BK(Ca) transfection in BK(Ca) non-expressing CHO cells. The changes in [Ca(2+)](i) were determined using a calcium-sensitive dye, fluo 3-AM. Membrane potential changes were detected with a potential-sensitive dye, DiBAC(4)(3). We found that laminar SS inhibited VSMC proliferation and stimulated BK(Ca) channel expression. Furthermore, laminar SS induced an increase in [Ca(2+)](i) and membrane hyperpolarization. Besides in VSMC, the inhibitory effect of BK(Ca) channel activity on cell proliferation in response to SS was also confirmed in BK(Ca)-transfected CHO cells showing a decline in proliferation. Blocking BK(Ca) channel reversed its inhibitory effect, providing additional support for the involvement of BK(Ca) in SS-induced proliferation reduction. Our results suggest, for the first time, that BK(Ca) channel mediates laminar SS-induced inhibition of VSMC proliferation. This finding is important for understanding the mechanism by which SS regulates VSMC proliferation, and should be helpful in developing strategies to prevent flow-initiated vascular disease formation.