Skeletal muscle contraction is triggered by acetylcholine induced release of Ca2+ from sarcoplasmic reticulum. Although this signaling pathway is independent of extracellular Ca2+, L-type voltage-gated calcium channel (Cav) blockers have inotropic effects on frog skeletal muscles which occur by an unknown mechanism. Taking into account that skeletal muscle fiber expresses Ca+2-sensitive adenylyl cyclase (AC) isoforms and that cAMP is able to increase skeletal muscle contraction force, we investigated the role of Ca2+ influx on mouse skeletal muscle contraction and the putative crosstalk between extracellular Ca2+ and intracellular cAMP signaling pathways. The effects of Cav blockers (verapamil and nifedipine) and extracellular Ca2+ chelator EGTA were evaluated on isometric contractility of mouse diaphragm muscle under direct electrical stimulus (supramaximal voltage, 2 ms, 0.1 Hz). Production of cAMP was evaluated by radiometric assay while Ca2+ transients were assessed by confocal microscopy using L6 cells loaded with fluo-4/AM. Ca2+ channel blockers verapamil and nifedipine had positive inotropic effect, which was mimicked by removal of extracellular Ca+2 with EGTA or Ca 2+-free Tyrode. While phosphodiesterase inhibitor IBMX potentiates verapamil positive inotropic effect, it was abolished by AC inhibitors SQ22536 and NYK80. Finally, the inotropic effect of verapamil was associated with increased intracellular cAMP content and mobilization of intracellular Ca 2+, indicating that positive inotropic effects of Ca2+ blockers depend on cAMP formation. Together, our results show that extracellular Ca2+ modulates skeletal muscle contraction, through inhibition of Ca2+-sensitive AC. The cross-talk between extracellular calcium and cAMP-dependent signaling pathways appears to regulate the extent of skeletal muscle contraction responses. © 2013 Elsevier B.V. All rights reserved.