We have studied the effects of mitochondria poisoning by carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) on Ca2+ signaling in enzymatically dissociated mouse flexor digitorum brevis (FDB) muscle fibers. We used Fura-2AM to measure resting [Ca2+]i and MagFluo-4AM to measure Ca2+ transients. Exposure to FCCP (2 μM, 2 min) caused a continuous increase in [Ca2+]i at a rate of 0.60 nM/s and a drastic reduction of electrically elicited Ca2+ transients without much effect on their decay phase. Half of the maximal effect occurred at [Ca2+]i = 220 nM. This effect was partially reversible after long recuperation and was not diminished by Tiron, a reactive oxygen species (ROS) scavenger. FCCP had no effects on fiber excitability as shown by the generation of action potentials. 4CmC, an agonist of ryanodine receptors, induced a massive Ca2+ release. FCCP diminished the rate but not the amount of Ca2+ released, indicating that depletion of Ca2+ stores did not cause the decrease in Ca2+ transient amplitude. Ca2+ transient amplitude could also be diminished, but to a lesser degree, by increases in [Ca2+]i induced by repetitive stimulation of fibers treated with ciclopiazonic acid. This suggests an important role for Ca2+ in the FCCP effect on transient amplitude.