We have cloned the cDNA encoding the voltage-dependent K + channel Kv2.1 from human brain (hKv2.1). RNase protection and RT-PCR (reverse transcriptase-PCR) experiments reveal abundant Kv2.1 transcripts in human brain with virtually no expression detectable in human heart. hKv2.1 has been stably transfected into a human glioblastoma cell line, and transformed cells display large, slowly activating outward currents. The kinetics, steady-state activation and inactivation parameters, and external tetraethylammonium sensitivity were all similar to those described previously for hKv2.1 channels transiently expressed in Xenopus oocytes or other mammalian cell lines. A number of dopamine receptor antagonist/antipsychotic agents were shown to block hKv2.1. Trifluoperizine, trifluperidol and pimozide produced time-dependent blockade of hKv2.1 with IC 5 0 values of approx. 1-2 μM. The diphenylbutylpiperidine fluspirilene was shown to be 4-5-fold more potent than the other agents tested inhibiting hKv2.1 current with an IC 5 0 value of 297 nM. The block produced by fluspirilene was both time- and frequency-dependent. Furthermore, fluspirilene (1 μM) shifted the midpotential of the hKv2.1 steady-state inactivation curve by approx. 15 mV in the hyperpolarizing direction. These results demonstrate the usefulness of this transfection system for the pharmacological characterization of hKv2.1. Fluspirilene proved to be a relatively potent blocker of hKv2.1 and may provide a useful starting point for the development of more potent and selective agents active against this brain K + channel.