Trimeric intracellular cation channels (TRIC-A and TRIC-B) are located in the sarcoplasmic reticulum (SR) of most cells and are present in both cardiac and skeletal muscle. Mutant mice lacking both TRIC-A and TRIC-B channels die due to embryonic heart failure demonstrating their essential, but as yet, uncharacterised role in the heart (1). Identifying the distinct biophysical properties of TRIC-A and TRIC-B channels derived from the SR of normal muscle is not possible due to the presence of both types of channel, yet this is crucial for a full understanding of their roles in cardiac excitation-contraction coupling. We have therefore isolated the light SR membrane (LSR) fraction from mouse TRIC-A knockout tissue. LSR vesicles were fused with planar phosphatidylethanolamine lipid bilayers as previously described (2) and single-channel recordings of native TRIC-B channels were obtained under voltage-clamp conditions in symmetrical solutions of 210 mM K-PIPES, pH 7.2. The LSR vesicles incorporated into the bilayers in a fixed orientation such that the cis chamber corresponded to the cytosolic side of the channels and the trans chamber corresponded with the luminal face of the channels. Using these isolation procedures and experimental recording conditions, we find that the single-channel conductance of TRIC-B channels varies between channels. The maximum (or full) open state that we observe, falls within the approximate range 170-230 pS (n=27). The TRIC-B channels always gate in sub-conductance states and while these are also of a variable nature, predominant sub-conductance levels are found at 158 ± 4 pS (n=17; S.E.M.), 122±1 pS (n=19; S.E.M.), 93 ± 2 pS (n=18; S.E.M.) and 62±2 pS (n=18; S.E.M.). TRIC-B channel gating was voltage-dependent and channels were inhibited at negative holding potentials. For example, open probability was 0.05±0.012 at +30 mV but only 0.005±0.003 at -30 mV (n=5; S.E.M.; student’s t-test *p<0.05). Application of 300 mM KCl to the cytosolic channel side produced a parallel shift in the current-voltage relationship and a shift in the reversal potential to approximately -20 mV indicating that TRIC-B is not permeable to anions. These results differ from our previous conductance measurements of recombinantly expressed, detergent purified TRIC-B channels which exhibited a maximum single-channel conductance of approximately 140 pS (2). This study demonstrates that the conductance and gating properties of TRIC-B channels are exceptionally labile and may be easily perturbed by detergent purification procedures and/or use of yeast expression systems. The intrinsic variability of TRIC-B channel gating may be an important regulatory feature which enables flexible physiological control over monovalent cation fluxes across the SR.