Long-lasting or “L-type” calcium (CaL) channels mediate voltage-dependent Ca2+ influx and contraction in vascular smooth muscle cells (VSMCs). We have suggested that pressure-induced depolarization of VSMCs induces the expression of the pore-forming α1C subunit of the CaL channel, thereby contributing to the elevated Ca2+ influx and vascular tone observed in arteries of hypertensive rats. This study investigated the hypothesis that CaL channel β subunits, which may increase the cell surface expression of CaL channels, promote CaL channel expression in the VSMCS of small renal arteries. Initial Western blots indicated that cultured rat renal arteries minimally expressed the β2 subunit, which was apparent as an immunoreactive band at ~68 kD in renal VSMC membranes. In similar renal arteries, incorporation of fluorescein-labeled antisense directed against the β subunit resulted in a profound loss of CaL channel α1C subunits. The downregulation of CaL channel α1C subunits in response to βantisense was associated with a loss of Ca2+-dependent contraction in renal arteries. Interestingly, depolarization of renal VSMCs for 48 hours resulted in an upregulation of the β2 subunit that correlated with an increased expression of CaL channel α1C subunit proteins. Thus, the expression of functional CaL channel α1C subunits in small arteries may rely on ancillary β subunits, which may promote the trafficking, insertion or membrane stability of the CaL channel in the VSMC membrane. Supported by NIH R01HL064806-07.