Ca²⁺ oscillations are one of the most important signals within the cell. The mechanism for generation of Ca²⁺ oscillations is still not yet fully elucidated. We studied the role of capacitative Ca²⁺ entry (CCE) on intracellular Ca²⁺ oscillations induced by testosterone at the single-cell level in primary myotubes. Testosterone (100 nM) rapidly induced an intracellular Ca²⁺ rise, accompanied by Ca²⁺ oscillations in a majority of myotubes. Spectral analysis of the Ca²⁺ oscillations revealed a periodicity of 20.3 ± 1.8 s (frequency of 49.3 ± 4.4 mHz). In Ca²⁺-free medium, an increase in intracellular Ca²⁺ was still observed, but no oscillations. Neither nifedipine nor ryanodine affected the testosterone-induced Ca²⁺ response. This intracellular Ca²⁺ release was previously shown in myotubes to be dependent on inositol-1,4,5-trisphosphate (IP₃). Intracellular Ca²⁺ store depletion in Ca²⁺-free medium, using a sarcoplasmic/endoplasmic reticulum calcium ATPase-pump inhibitor, followed by re-addition of extracellular Ca²⁺, gave a fast rise in intracellular Ca²⁺, indicating that CCE was present in these myotubes. Application of either testosterone or albumin-bound testosterone induced Ca²⁺ release and led to CCE after re-addition of Ca²⁺ to Ca²⁺-free extracellular medium. The CCE blockers 2-aminoethyl diphenylborate and La³⁺, as well as perturbation of the cytoskeleton by cytochalasin D, inhibited testosterone-induced Ca²⁺ oscillations and CCE. The steady increase in Ca²⁺ induced by testosterone was not, however, affected by either La³⁺ or cytochalasin D. These results demonstrate testosterone-induced Ca²⁺ oscillations in myotubes, mediated by the interplay of IP₃-sensitive Ca²⁺ stores and Ca²⁺ influx through CCE.