We report here the expression in C2C12 myoblasts of the intermediate-conductance Ca²⁺-activated K⁺ (IK_Ca) channel. The IK_Ca current, recorded under perforated-patch configuration, had a transient time course when activated by ionomycin (0.5 µM; peak current density 26.2 ± 3.7 pA/pF, n = 10), but ionomycin (0.5 µM) plus DC-EBIO (100 µM) evoked a stable outward current (28.4 ± 8.2 pA/pF, n = 11). The current was fully inhibited by charybdotoxin (200 nM), clotrimazole (2 µM), and NPPB (300 µM), but not by TEA (1 mM) or d-tubocurarine (300 µM). Congruent with the IK_Ca channel, elevation of intracellular Ca²⁺ to inside-out patches resulted in the activation of a voltage-insensitive K⁺ channel with weak inward rectification, a unitary conductance of 38 ± 6 pS (at negative voltages), and a IC₅₀ for Ca²⁺ of 530 nM. The IK_Ca channel was activated metabotropically by external application of ATP (100 µM), an intracellular Ca²⁺ mobilizer. Under current clamp conditions, ATP application resulted in a membrane hyperpolarization of ~35 mV. The IK_Ca current downregulated during myogenesis, ceasing to be detectable four days following placing the myoblasts in differentiating medium. Downregulation was prevented by the myogenic suppressor agent bFGF. We also found that block of the IK_Ca channel by charybdotoxin did not inhibit bFGF-sustained myoblasts proliferation. These observations show that in C2C12 myoblasts the IK_Ca channel expression correlates inversely with differentiation, yet it does not appear to have a role in myoblast proliferation.