The effect of calcium-phospholipid-dependent protein kinase (PKC) activation on neurosteroid modulation of the GABA(A) receptor was examined in Xenopus oocytes expressing human recombinant alpha1beta2gamma2L GABA(A) receptors. GABA-gated chloride currents were measured using the two-electrode voltage-clamp technique. The peak amplitude of GABA-gated chloride currents was reduced by the PKC activator phorbol 12-myristate 13-acetate (PMA), but not by the inactive analog phorbol 12-mono-myristate (PMM). This effect of PMA was inhibited by the protein kinase inhibitor staurosporine. To investigate whether the activation of PKC could alter neurosteroid modulation of the GABA(A) receptor, the effect of PMA was studied on the positive allosteric modulatory steroid 3alpha,21-dihydroxy-5alpha-pregnan-20-one (THDOC) and the negative modulatory neurosteroid pregnenolone sulfate (PS). THDOC potentiation of GABA-gated chloride currents was found to be increased by approximately 120% following PMA treatment, while PS inhibition was not affected. The increase in THDOC potentiation by PMA was blocked by staurosporine. No change in THDOC potentiation was observed following PMM treatment. The enhancement of THDOC potentiation following PMA treatment was not due to a shift in the GABA EC50. In addition to inhibiting the peak amplitude of the GABA response, PMA treatment resulted in non-desensitizing GABA responses. Similarly, GABA responses of receptors which had been desensitized with prolonged GABA application also showed a reduction in peak amplitude and reduced desensitization. THDOC potentiation of desensitized receptors was enhanced approximately 70% with respect to non-desensitized receptors. The present results demonstrate that protein phosphorylation and receptor desensitization alter modulation of the GABA(A) receptor complex by some neurosteroids.