In the adult brain, chloride (Cl−) influx through GABAAreceptors is an important mechanism of synaptic inhibition. However, under a variety of circumstances, including acquired epilepsy, neuropathic pain, after trains of action potentials or trauma, and during normal early brain development, GABAAreceptor activation excites neurons by gating Cl−efflux because the intracellular Cl−concentration (Cli) is elevated. These findings require an inducible, active mechanism of chloride accumulation. We used gramicidin-perforated patch recordings to characterize Cl−transport via NKCC1, the principal neuronal Cl−accumulator, in neonatal CA1 pyramidal neurons. NKCC1 activity was required to maintain elevated Clisuch that GABAAreceptor activation was depolarizing. Kinetic analysis of NKCC1 revealed reversible transmembrane Cl−transport characterized by a large maximum velocity (vmax) and high affinity (Km), so that NKCC1 transport was limited only by the net electrochemical driving force for Na+, K+, and Cl−. At the steady-state Cli, NKCC1 was at thermodynamic equilibrium, and there was no evidence of net Cl−transport. Trains of action potentials that have been previously shown to induce persistent changes in neuronalECl(reversal potential for Cl−) did not altervmaxorKmof NKCC1. Rather, action potentials shifted the thermodynamic set point, the steady-state Cliat which there was no net NKCC1-mediated Cl−transport. The persistent increase in Clirequired intact α2/α3 Na+-K+-ATPase activity, indicating that trains of action potentials reset the thermodynamic equilibrium for NKCC1 transport by lowering Nai. Activity-induced changes in Na+-K+-ATPase activity comprise a novel mechanism for persistent alterations in synaptic signaling mediated by GABA.