We previously showed that shrinking a barnacle muscle fiber (BMF) in a hypertonic solution (1,600 mosM/kg) stimulates an amiloride-sensitive Na-H exchanger. This activation is mediated by a G protein and requires intracellular Cl-. The purpose of the present study was to determine (a) whether Cl- plays a role in the activation of Na-H exchange under normotonic conditions (975 mosM/kg), (b) the dose dependence of [Cl-]i for activation of the exchanger under both normo- and hypertonic conditions, and (c) the relative order of the Cl-- and G-protein-dependent steps. We acid loaded BMFs by internally dialyzing them with a pH-6.5 dialysis fluid containing no Na+ and 0-194 mM Cl-. The artificial seawater bathing the BMF initially contained no Na+. After dialysis was halted, adding 50 mM Na+ to the artificial seawater caused an amiloride-sensitive pHi increase under both normo- and hypertonic conditions. The computed Na-H exchange flux (JNa-H) increased with increasing [Cl-]i under both normo- and hypertonic conditions, with similar apparent Km values ( approximately 120 mM). However, the maximal JNa-H increased by nearly 90% under hypertonic conditions. Thus, activation of Na-H exchange at low pHi requires Cl- under both normo- and hypertonic conditions, but at any given [Cl-]i, JNa-H is greater under hyper- than normotonic conditions. We conclude that an increase in [Cl-]i is not the primary shrinkage signal, but may act as an auxiliary shrinkage signal. To determine whether the Cl--dependent step is after the G-protein-dependent step, we predialyzed BMFs to a Cl--free state, and then attempted to stimulate Na-H exchange by activating a G protein. We found that, even in the absence of Cl-, dialyzing with GTPgammaS or AlF3, or injecting cholera toxin, stimulates Na-H exchange. Because Na-H exchange activity was absent in control Cl--depleted fibers, the Cl--dependent step is at or before the G protein in the shrinkage signal-transduction pathway. The stimulation by AlF3 indicates that the G protein is a heterotrimeric G protein.