Sarcoplasmic reticulum (SR) K+ channels are voltage-sensitive, monovalent cation channels present in high levels throughout the junctional and longitudinal SR of cardiac and skeletal muscle. They are suggested to equilibrate K+ concentrations and balance charge movements across the SR membrane during Ca2+-release and reuptake (1, 2). The novel proteins, TRIC-A and TRIC-B, are thought to represent two subtypes of SR K+ channel (3, 4). To study the gating of a homogeneous population of SR K+ channels, we incorporated SR membrane vesicles from TRIC-A KO mice into artificial membranes under voltage-clamp conditions in symmetrical 210 mM K+ PIPES solutions as previously described (4, 5). SR vesicles incorporated in a fixed orientation and the cytosolic side of the channels was voltage-clamped at potentials relative to the luminal side, which was held at ground. When multiple channels were observed, we added decamethonium to the cytosolic chamber at the end of the experiment to enable us to count the channels in the bilayer and determine the zero current level. Mean values ± SEM were compared by Student’s t test. When only one SR K+ channel was gating in the bilayer, the gating was obviously voltage-dependent; Po was 0.099±0.014 (n=35) at +30 mV and 0.007±0.004 (n=19) at -30 mV (p<0.001). At +30mV, the average SR K+ channel Po increased to 0.251±0.046 (n=12) when there were two channels, 0.493±0.067 (n=6) when there were three channels, and 0.591±0.06 (n=3) when there were four channels gating in the bilayer (p<0.001). A similar trend was observed at -30 mV, thus indicating that the channels are behaving in a positively cooperative manner at both holding potentials. The experimental data deviated from the binomial statistical prediction that would be expected if identical, independent SR K+ channels were gating simultaneously, further supporting the idea of cooperative gating. We also investigated whether the presence of ryanodine receptor (RyR) channels gating in the bilayer would influence the activity of the SR K+ channels. Most SR vesicle fusions led to incorporation of only SR K+ channels into the bilayers, however, when an RyR channel incorporated also, this did not appear to influence the gating of the SR K+ channels. With an RyR also present in the bilayer, SR K+ channel Po was 0.130±0.041 at +30 mV (n=10) and 0.030±0.024 at -30 mV (n=5), similar to SR K+ channel Po without an accompanying RyR. The cooperative gating feature of SR K+ channels that we observe may be central to their ability to rapidly respond to signals such as small voltage changes across the SR, thus enabling large SR K+ fluxes to rapidly compensate K+ gradients or charge differences across the SR. Future work is necessary to identify the mechanisms underlying the cooperativity and to determine whether any SR K+ channel modulators could directly affect this behaviour.