It was hypothesized that the caffeine derivative paraxanthine results in subcontracture increases in intracellular calcium concentration ([Ca2+]i) in resting skeletal muscle. Single fibers obtained from mouse flexor digitorum brevis were loaded with a fluorescent Ca2+ indicator, indo 1-acetoxymethyl ester. After a stable baseline was recorded, the fiber was superfused with physiological salt solution (Tyrode) containing 0.5, 1.0, 2.5, or 5 mM paraxanthine, resulting in [Ca2+]iincreases of 6.4 ± 2.5, 9.7 ± 3.6, 26.8 ± 11.7, and 39.6 ± 9.6 nM, respectively. The increases in [Ca2+]i were transient and were also observed with exposure to 5 mM theophylline and theobromine. Six fibers were exposed to 5 mM paraxanthine followed by 5 mM paraxanthine in the presence of 10 mM procaine (sarcoplasmic reticulum Ca2+release channel blocker). There was no increase from baseline [Ca2+]i when fibers were superfused with paraxanthine and procaine, suggesting that the sarcoplasmic reticulum is the primary Ca2+ source in the paraxanthine-induced response. In separate experiments, intact flexor digitorum brevis ( n = 13) loaded with indo 1-acetoxymethyl ester had a significant increase in [Ca2+]i with exposure to 0.01 mM paraxanthine. It is concluded that physiological and low pharmacological concentrations of paraxanthine result in transient, subcontracture increases in [Ca2+]i in resting skeletal muscle, the magnitude of which is related to paraxanthine concentration.