This study describes the pharmacological properties of GABAA receptors expressed in P19-N cells using fluorescence imaging of intracellular chloride with 6-methoxy-N-ethylquinolinium iodide (MEQ). We show that application of the GABA agonist, muscimol (10-200 microM), produces time- and concentration-dependent increases in intracellular [Cl-] that are blocked by bicuculline. Diazepam (10 microM) and pentobarbital (1 mM) potentiate muscimol-stimulation. These receptors exhibit novel pharmacological properties. The neurosteroid, 3alpha-hydroxy-5alpha-pregnane-20-one (1-10 microM) exhibited weak potency in enhancement of muscimol-stimulation. Ethanol (50 and 100 mM) exhibited high efficacy on muscimol responses, a 4- to 5-fold potentiation, respectively, of muscimol (10 microM) alone. GABA and muscimol allosterically modulated specific binding of [3H]flunitrazepam to differentiated P19 cells. Modulation of GABAA receptor mediated increases in intracellular [Cl-] demonstrated stability in response magnitude from 7 to 15 days following removal of retinoic acid. In concert, GABAA receptor subunit mRNA and protein expression patterns in these neuron-like cells were stable over the same period. Using RT-PCR we determined that differentiated P19 cells lack gamma1, gamma2L, alpha6 and delta subunit mRNAs while expressing alpha1, alpha2, alpha3, alpha4, alpha5, beta1, beta2, beta3, gamma2S and gamma3. Furthermore, subunit specific antibody immunocytochemical labeling of cells with a neuronal morphology indicated the presence of alpha1, alpha2, alpha4, and gamma2 subunits (the only subunits tested). Therefore, P19-N cells should prove useful to researchers in need of a model cell culture system in which to study function and regulation of neuronal GABAA receptors.