Inhibitory GABAergic synaptic transmission in the mammalian hippocampus depends upon a hyperpolarized reversal potential for Cl − (E Cl ). To examine the regulation of E Cl hyperpolarization we cultured hippocampal neurons for two weeks in either a low- or a high-concentration of KCl (2.6 or 18.7 mM, respectively). Neurons were then recorded from standard extracellular solution containing 3 mM K + , using the dual perforated patch clamp technique. Low-KCl cultured neurons fired spontaneous action potentials (APs; 0.33±0.11 Hz), while high-KCl cultured neurons were quiescent, resulting in a significant difference in AP activity (p=0.042). This high-KCl-induced decrease in activity was accompanied by depolarizations of both the AP threshold (p<0.001) and E Cl (p<0.001), and a decrease in input resistance (IR, p<0.001), when compared with low-KCl cultured neurons. Blocking AP firing of low-KCl neurons during culturing with 1 μM tetrodotoxin did not alter E Cl hyperpolarization, when compared with drug-free cultured low-KCl neurons (p=0.627); thus AP firing is not required for E Cl hyperpolarization. Acute perfusion of a high-KCl extracellular solution onto low- or high-KCl cultured neurons demonstrated that high-KCl significantly depolarized the resting membrane potential (RMP). The KCl-induced change in E Cl did not correspond with alterations in the expression of the cation chloride cotransporters KCC2 and NKCC1, as determined by western blotting (p=0.736). These findings suggest that: (1) extracellular K + regulates E Cl hyperpolarization; and, (2) the use of high-KCl during neuronal culture produces biophysically abnormal parameters, and thus should be discouraged.