The desensitization of alpha-bungarotoxin-insensitive native neuronal nicotinic receptors was studied in rat cortical cell cultures using the patch clamp technique. Thirty-minute perfusions of nicotine reduced currents evoked by short test pulses of 300 microM acetylcholine over a range of 3 to 300 nM, with an IC50 of 51 nM. The time course of desensitization onset was fit by a biexponential function consisting of a fast time constant of about 1 min and a slower component of 6-10 min. The desensitization recovery process was also biexponential and was dominated by a slow time constant of 12-20 min, as well as a minor component of about 1 min. The intracellular dialysis of either the protein kinase C activator phorbol-12-myristate-13 acetate or the phosphatase inhibitor cyclosporin A accelerated the desensitization recovery rate by 2-fold. The data imply that endogenous cortical nicotinic receptor channels may enter one of two desensitization states. The first state (D1) is characterized by rapid entry and recovery, whereas transitions into and out of the second state (D2) occur at slower rates. The D2 receptor state may arise by a sequential transition from the D1 conformation. Protein kinase C activation or phosphatase 2B inhibition may favor the D1 receptor state over that of D2 to promote faster overall rates of desensitization recovery.