Ca²⁺ release from the intracellular store, the sarcoplasmic reticulum, is largely mediated by two receptor channel complexes, the ryanodine receptor (RYR) and the IP₃ receptor (IP₃R). A family of FK506-binding proteins (FKBPs) and a Ca²⁺-dependent phosphatase, calcineurin, may each interact with both RYR and IP₃R to modulate receptor-mediated Ca²⁺ release. The physiological effects of FKBPs and calcineurin on RYR and IP₃-mediated Ca²⁺ release were each investigated in single voltage-clamped smooth muscle cells which were isolated from the colon of guinea-pigs (humanely killed by cervical dislocation and immediate exsanguination). [Ca²⁺]_c was measured as fluorescence using the membrane-impermeable dye fluo 3 (penta-ammonium salt) introduced into the cell via the patch pipette. IP₃ (25 µM) was released from its caged compound by flash photolysis and caffeine (10 mM) was applied by hydrostatic pressure ejection. The FK506binding protein 12 kDa (FKBP12) and calcineurin were confirmed to be associated with RYR2 and IP₃R by co-immunoprecipitation. The immunosuppressant FK506 (20 µM) did not affect the extent of the association between calcineurin and IP₃R. FK506 (10 µM, which dissociates FKBPs and inhibits calcineurin) increased the [Ca²⁺]_c rise each evoked by the RYR activator caffeine and by IP₃R activation following photolysed caged IP₃.Another immunosuppressant rapamycin (10 µM, which dissociates FKBPs but does not inhibit calcineurin) also increased the amplitude of the caffeine-evoked but reduced that of the IP₃-evoked [Ca²⁺]_c transient. The calcineurin inhibitors, cypermethrin (10 µM) and okadaic acid (5 µM) each increased the IP₃-evoked [Ca²⁺]_c transient. Cypermethrin also increased the [Ca²⁺]_c rise evoked by caffeine. Following inhibition of calcineurin by cypermethrin, FK506 reduced the IP₃-evoked [Ca²⁺]_c transient in contrast to the increase occurring in the absence of cypermethrin. Together, these results indicate that FKBP12 binds to RYR2 and reduces its activity in smooth muscle but potentiates IP₃R activity. Calcineurin regulates both RYR and IP₃R by reducing channel activity.