We previously showed that plasma membrane Ca²⁺-ATPase (PMCA) activity accounted for 25-30% of relaxation in bladder smooth muscle (8). Among the four PMCA isoforms only PMCA1 and PMCA4 are expressed in smooth muscle. To address the role of these isoforms, we measured cytosolic [Ca²⁺] using Fura-PE3 and simultaneously measured contractility in bladder smooth muscle from wild-type (WT), Pmca1^+/-, Pmca4^+/-, Pmca4^-/- and Pmca1^+/-Pmca4^-/- mice. There were no differences in basal [Ca²⁺]_i values between bladder preparations. KCl (80 mM) elicited both larger forces (150-190%) and increases in cytosolic Ca²⁺ (130-180%) in smooth muscle from Pmca1^+/- and Pmca1^+/-Pmca4^-/- bladders than those in WT or Pmca4^-/-. The responses to carbachol (CCh: 10 µM) were also greater in Pmca1^+/- (120-150%) than in WT bladders. In contrast, the responses in Pmca4^-/- and Pmca1^+/-Pmca4^-/- bladders to CCh were significantly smaller (40-50%) than WT. The rise half-times of force and [Ca²⁺]_i increases in response to KCl and CCh and the concomitant half-times of their decrease upon washout of agonist were prolonged in Pmca4^-/- (130-190%) and Pmca1^+/-Pmca4^-/- (120-250%) bladders, but not in Pmca1^+/- bladders with respect to WT. Our evidence indicates distinct isoform functions with the PMCA1 isoform involved in overall Ca²⁺-clearance, while PMCA4 is essential for the [Ca²⁺]_i increase and contractile response to the CCh receptor-mediated signal transduction pathway.