The effects of ciprofloxacin on mitochondrial DNA (mtDNA) content, oxygen consumption, mitochondrial membrane potential, cellular ATP formation, and capacitative Ca2+ entry into Jurkat cells were investigated. In cells incubated for several days with 25 μg/ml ciprofloxacin, a 60% reduction of mtDNA content, inhibition of the respiratory chain, and a significant decrease in mitochondrial membrane potential were observed. These changes led to a decrease in the calcium buffering capacity of mitochondria which, in turn, resulted in a gradual inhibition of the capacitative Ca2+ entry. On days 4, 7, and 11 of incubation with ciprofloxacin, the initial rates of Ca2+ entry were reduced by 33%, 50%, and 50%, respectively. Ciprofloxacin caused a transient decrease in the cellular capability for ATP formation. In cells incubated for 15 min with glucose, pyruvate, and glutamine as exogenous fuel, ciprofloxacin reduced ATP content by 16% and 35% on days 4 and 7, respectively, of incubation with the drug. However, on day 11 of incubation with ciprofloxacin, a recovery of cellular ATP formation was observed. In conclusion, long-term exposure of Jurkat cells to ciprofloxacin at a concentration of 25 μg/ml seriously affects cellular energy metabolism and calcium homeostasis.