FKBP12 and FKBP12.6 form part of the macromolecular ryanodine receptor (RyR) complex in striated muscle and there is general agreement that dissociation of FKBPs from RyRs is involved in the progression of heart disease and skeletal muscle disorders. There is high structural homology between FKBP12 and FKBP12.6 and evidence suggests that both proteins compete for the same binding sites on the cardiac isoform of RyR (RyR2) (1). However, whereas FKBP12 activates RyR2, FKBP12.6 has negligible ability as an activator of RyR2 and behaves instead as an antagonist of FKBP12 (1). To investigate the structural differences between FKBP12 and FKBP12.6 that are important for conferring the ability to activate RyR2, we generated a triple mutant of FKBP12 (FKBP12E31Q/D32N/W59F), where the amino acids Glu31, Asp32 and Trp59 were mutated to the corresponding residues in FKBP12.6 (Gln31, Asn32 and Phe59). FKBP12E31Q/D32N/W59F was cloned and recombinantly expressed in E.coli. The effects of FKBP12, FKBP12.6 and FKBP12E31Q/D32N/W59F on the gating of single cardiac (RyR2) and skeletal (RyR1) channels incorporated into planar phosphatidylethanolamine bilayers were compared using previously described methods (1). FKBP12E31Q/D32N/W59F had no effect on RyR2 channel gating (open probability (Po) was 0.206±0.08 before, and 0.223±0.10 after, addition of 1 µM FKBP12E31Q/D32N/W59F (SEM; n=5)). The triple FKBP12 mutant, therefore, lost the ability to activate RyR2 and instead behaved more like FKBP12.6. Further experiments were carried out with RyR1 and we found that FKBP12 significantly inhibited RyR1 whereas both FKBP12.6 and FKBP12E31Q/D32N/W59F were activators (Po was 0.021±0.008 before, and 0.110±0.026 after, 1 µM FKBP12E31Q/D32N/W59F (SEM; n=7; p<0.01)). In summary, our results demonstrate a clear distinction in the regulatory effects of FKBPs on RyR1 and RyR2. We also demonstrate that, following the binding of FKBP12/FKBP12.6 to RyR1 or RyR2, the capacity to functionally regulate channel gating is contained within the amino acid residues Glu31, Asp32 and Trp59 in FKBP12 and Gln31, Asn32 and Phe59 in FKBP12.6. These findings are relevant to understanding physiological regulation of sarcoplasmic reticulum Ca2+ release and the role of FKBPs in cardiac and skeletal muscle disease.