Ca2+ is known to enter skeletal muscle at rest and during activity. Except for the well-characterized Ca2+ entry through L-type channels, pathways involved in these Ca2+ entries remain elusive in adult muscle. This study investigates Ca2+ influx at rest and during activity using the method of Mn2+ quenching of fura-2 fluorescence on voltage-controlled adult skeletal muscle cells. Resting rate of Mn2+ influx depended on external [Mn2+] and membrane potential. At -80 mV, replacement of Mg2+ by Mn2+ gave rise to an outward current associated with an increase in cell input resistance. Calibration of fura-2 response indicated that Mn2+ influx was too small to be resolved as a macroscopic current. Partial depletion of the sarcoplasmic reticulum induced by a train of action potentials in the presence of cyclopiazonic acid led to a slight increase in resting Mn2+ influx but no change in cell input resistance and membrane potential. Trains of action potentials considerably increased Mn2+ entry through an electrically silent pathway independent of L-type channels, which provided 24% of the global Mn2+ influx at +30 mV under voltage-clamp conditions. Within this context, the nature and the physiological role of the Ca2+ pathways involved during muscle excitation still remain open questions.