During electrical activity of skeletal muscle, K⁺ accumulates in the T-tubules. The expected after-depolarization is largely prevented by Cl^-conducted by CLC channels. Therefore, normal skeletal muscle shows no action potential (AP) bursts in response to a single stimulus in contrast to myotonic muscle which is hyperexcitable due to a reduced Cl- conductance. We tested whether deficiency of Ca²⁺-activated large conductance K⁺ channels (BK channels) prevents the generation of myotonia by the CLC blocker 9-anthracene carboxylic acid (9-AC, 50 mM). We used excised EDL and soleus muscles from BK-/- and wild-type (WT) mice and measured the muscle relaxation time of single twitches elicited by supramaximal stimuli. In some WT muscle, BK channels were blocked with 5 mM paxilline. Whereas myotonia was generated by 9-AC in normal muscle, myotonia could not be evoked in BK-deficient or BK-blocked EDL muscle. In the slow-twitching soleus muscle, the response was less clear. Immunohistochemistry showed BK channels in close vicinity of dihydropyridine and ryanodine receptors (DHPR, RYR). From our results it can be hypothesized: i) T-tubular activation of BK channels by local Ca²⁺ elevations via DHPR and/or RYR; ii) that K⁺ is conducted by BK channels during the repolarizing phase of an AP and involved in the genesis of T-tubular K⁺ accumulation.