The cellular and molecular processes underlying the regulation of ryanodine receptor (RyR) Ca2+ release in smooth muscle cells (SMCs) are incompletely understood. Here we show that FKBP12.6 proteins are expressed in pulmonary artery (PA) smooth muscle and associated with type-2 RyRs (RyR2), but not RyR1, RyR3, or IP3 receptors (IP3Rs) in PA sarcoplasmic reticulum. Application of FK506, which binds to FKBPs and dissociates these proteins from RyRs, induced an increase in [Ca2+]i and Ca2+-activated Cl− and K+ currents in freshly isolated PASMCs, whereas cyclosporin, an agent known to inhibit calcineurin but not to interact with FKBPs, failed to induce an increase in [Ca2+]i. FK506-induced [Ca2+]i increase was completely blocked by the RyR antagonist ruthenium red and ryanodine, but not the IP3R antagonist heparin. Hypoxic Ca2+ response and hypoxic vasoconstriction were significantly enhanced in FKBP12.6 knockout mouse PASMCs. FK506 or rapamycin pretreatment also enhanced hypoxic increase [Ca2+]i, but did not alter caffeine-induced Ca2+ release (SR Ca2+ content) in PASMCs. Norepinephrine-induced Ca2+ release and force generation were also markedly enhanced in PASMCs from FKBP12.6 null mice. These findings suggest that FKBP12.6 plays an important role in hypoxia- and neurotransmitter-induced Ca2+ and contractile responses by regulating the activity of RyRs in PASMCs.