The myoplasmic free Ca²⁺concentration ([Ca²⁺]_i) was measured in intact single fibers from mouse skeletal muscle with the fluorescent Ca²⁺ indicator indo 1. Some fibers were perfused in a solution in which the concentration of Na⁺ was reduced from 145.4 to 0.4 mM (low-Na⁺solution) in an attempt to activate reverse-mode Na⁺/Ca²⁺exchange (Ca²⁺ entry in exchange for Na⁺ leaving the cell). Under normal resting conditions, application of low-Na⁺ solution only increased [Ca²⁺]_iby 5.8 ± 1.8 nM from a mean resting [Ca²⁺]_iof 42 nM. In other fibers, [Ca²⁺]_iwas elevated by stimulating sarcoplasmic reticulum (SR) Ca²⁺ release with caffeine (10 mM) and by inhibiting SR Ca²⁺ uptake with 2,5-di(tert-butyl)-1,4-benzohydroquinone (TBQ; 0.5 μM) in an attempt to activate forward-mode Na⁺/Ca²⁺exchange (Ca²⁺ removal from the cell in exchange for Na⁺ influx). These two agents caused a large increase in [Ca²⁺]_i, which then declined to a plateau level approximately twice the baseline [Ca²⁺]_iover 20 min. If the cell was allowed to recover between exposures to caffeine and TBQ in a solution in which Ca²⁺ had been removed, the increase in [Ca²⁺]_iduring the second exposure was very low, suggesting that Ca²⁺ had left the cell during the initial exposure. Application of caffeine and TBQ to a preparation in low-Na⁺ solution produced a large, sustained increase in [Ca²⁺]_iof ∼1 μM. However, when cells were exposed to caffeine and TBQ in a low-Na⁺ solution in which Ca²⁺ had been removed, a sustained increase in [Ca²⁺]_iwas not observed, although [Ca²⁺]_iremained higher and declined slower than in normal Na⁺ solution. This suggests that forward-mode Na⁺/Ca²⁺exchange contributed to the fall of [Ca²⁺]_iin normal Na⁺ solution, but when extracellular Na⁺ was low, a prolonged elevation of [Ca²⁺]_icould activate reverse-mode Na⁺/Ca²⁺exchange. The results provide evidence that skeletal muscle fibers possess a Na⁺/Ca²⁺exchange mechanism that becomes active in its forward mode when [Ca²⁺]_iis increased to levels similar to that obtained during contraction.