We have characterised the functional regulation of the type-3 ryanodine receptor by the 12 kDa FK506-binding protein. Wild-type type-3 ryanodine receptor and mutant type-3 ryanodine receptor in which the critical valine at position 2322 in the central 12 kDa FK506-binding protein binding site was substituted by aspartate, were stably expressed in human embryonic kidney cells. In contrast to the wild-type receptor, the mutant receptor was strongly impaired in binding to immobilised glutathione S-transferase 12 kDa FK506-binding protein. Caffeine-induced 45Ca2+-efflux was markedly increased in cells expressing mutant type-3 ryanodine receptor whereas the maximal-releasable Ca2+ was not affected. Confocal Ca2+ imaging provided clear evidence for a much higher sensitivity of the mutant receptor, which showed global Ca2+ release at about 20-fold lower caffeine concentrations than the wild-type receptor. Spontaneous Ca2+ sparks were observed in both wild-type- and mutant-expressing cells but the number of sparking cells was about 1.5-fold higher in the mutant group, suggesting that the degree of FK506 binding controls the stability of the closed state of ryanodine receptor channels. Furthermore, overexpression of 12 kDa FK506-binding protein decreased the number of sparking cells in the wild-type-expressing cells whereas it did not affect the number of sparking cells in cells expressing the mutant receptor. Concerning spark properties, the amplitude and duration of Ca2+ sparks mediated by mutant channels were significantly reduced in comparison to wild-type channels. This suggests that functional coupling between different mutant type-3 ryanodine receptor channels in a cluster is impaired. Our findings show for the first time that the central binding site for the 12 kDa FK506-binding protein of type-3 ryanodine receptor, encompassing the critical valine proline motif, plays a crucial role in the modulation of the Ca2+ release properties of the type-3 ryanodine receptor channel, including the regulation of both global Ca2+ responses and spontaneous Ca2+ sparks.