Ca2+ sparks, localized elevations in cytosolic [Ca2+], are rarely detected in intact adult mammalian skeletal muscle under physiological conditions. However, they have been observed in permeabilized cells and in intact fibres subjected to stresses, such as osmotic shock and strenuous exercise. Our previous studies indicated that an excess in cellular reactive oxygen species (ROS) generation over the ROS scavenging capabilities could be one of the up-stream causes of Ca2+ spark appearance in permeabilized muscle fibres. Here we tested whether the cytosolic ROS balance is compromised in intact skeletal muscle fibres that underwent osmotic shock and whether this misbalance contributes to unmasking Ca2+ sparks. Spontaneous Ca2+ sparks and the rate of ROS generation were assessed with single photon confocal microscopy and fluorescent indicators fluo-4, CM-H2DCFDA and MitoSOX Red. Osmotic shock produced spontaneous Ca2+ sparks and a concomitant significant increase in ROS production. Preincubation of muscle cells with ROS scavengers (e.g. MnTBAP, Mn-cpx 3, TIRON) nearly eliminated Ca2+ sparks. In addition, inhibitors of NAD(P)H oxidase (DPI and apocynin) significantly reduced ROS production and suppressed the appearance of Ca2+ sparks. Taken together, the data suggest that ROS contribute to the abnormal Ca2+ spark activity in mammalian skeletal muscle subjected to osmotic stress and also indicate that NAD(P)H oxidase is a possible source of ROS. We propose that ROS-dependent Ca2+ sparks are an important component of adaptive/maladaptive muscle responses under various pathological conditions such as eccentric stretch, osmotic changes during ischaemia and reperfusion, and some muscle diseases.