Transgenic mice lacking calcium channel beta3 subunits (CaVbeta3) were used to determine the involvement of a multimeric calcium channel in mediating stimulated renal calcium absorption. We measured the ability of calcium channel beta3 subunit-null (CaVbeta3-/-) and wild-type (CaVbeta3+/+) mice to increase renal calcium absorption in response to the calcium-sparing diuretic chlorothiazide (CTZ). Control rates of fractional sodium excretion were comparable in CaVbeta3-/- and CaVbeta3+/+ mice and CTZ increased sodium excretion similarly in both groups. CTZ enhanced calcium absorption only in wild-type CaVbeta3+/+ mice. This effect was specific for diuretics acting on distal tubules because both CaVbeta3-/- and CaVbeta3+/+ mice responded comparably to furosemide. The absence of beta3 subunits resulted in compensatory increases of TrpV5 calcium channels, the plasma membrane Ca-ATPase, NCX1 Na/Ca exchanger protein, and calbindin-D9k but not calbindin-D28k. We conclude that TrpV5 mediates basal renal calcium absorption and that a multimeric calcium channel that includes CaVbeta3 mediates stimulated calcium transport.