It is widely believed that, in situ, the FK506-binding protein, FKBP12.6, binds preferentially to the cardiac isoform of the ryanodine receptor (RyR2) (1) whereas FKBP12 normally binds to the skeletal isoform (RyR1) (2). However, although the physiological levels of FKBP12 (1-3 µM) are thought to be higher than those of FKBP12.6 (1), both binding proteins are present in cardiac and skeletal muscle cells. Few studies have investigated whether FKBP12 can modulate RyR2 function or whether FKBP12.6 can affect the activity of RyR1. We have therefore studied how FKBP12 and FKBP12.6 influence the gating of single native RyR1 and RyR2 channels. Rabbit RyR1 or sheep RyR2 were incorporated into planar phosphatidylethanolamine lipid bilayers under voltage-clamp conditions as previously described (3). Single-channel recordings were obtained with 250 mM Hepes, 80 mM Tris, 15 mM free Ca2+, pH 7.2, on the cis (cytoplasmic) side and 250 mM glutamic acid, 10 mM Hepes, pH to 7.2 with Ca(OH)2 (free [Ca2+] approximately 50 mM) on the trans (luminal) side of the bilayer. The luminal chamber was voltage-clamped at ground. Open probability (Po) was determined over 3 min of continuous recording. Student’s t-test was used to assess the difference between mean values. Cytosolic addition of 500 nM FKBP12 significantly decreased the Po of RyR1 from 0.021 ± 0.005 (S.E.M.; n = 4) in controls to 0.001 ± 0.001 (S.E.M.; n = 4; P<0.01). This effect was irreversible after perfusing away the FKBP12. In contrast, 200 nM FKBP12.6 increased RyR1 Po from 0.005 ± 0.002 to 0.156 ± 0.099 (S.E.M.; n = 13; P<0.05), again in an irreversible manner. Different effects were observed with RyR2. FKBP12 was a reversible activator of RyR2 (for example, 500 nM FKBP12 increased the Po from 0.190 ± 0.051 to 0.498 ± 0.141; S.E.M.; n = 14; P<0.05) whereas FKBP12.6, while displaying no intrinsic action itself could antagonise FKBP12-induced activation. It has been reported that FK-binding proteins are important for preventing sub-conductance state gating of RyR1 and RyR2. However, our experiments provide no evidence to suggest that the presence of FK-binding proteins can influence the incidence of sub-conductance states in either isoform. We did not observe clearly resolvable RyR2 sub-conductance gating in the presence or absence of FKBP12 or FKBP12.6. Although rare, brief sub-conductance state gating was characteristic of RyR1 but the incidence was not altered by adding FKBP12 or FKBP12.6. Since FK-binding proteins exert opposing actions on the two isoforms of RyR, the ratio of FKBP12/FKBP12.6 in the cytoplasm of cardiac and skeletal muscle cells will be critical for determining RyR activity in situ. Changes in this ratio may be crucial in heart failure where it has been suggested that RyR/FKBP interactions are altered (4).