Experiments were performed to compare the mechanism of block of voltage-dependent K channels by various short and long alkyl chain tetraalkylammonium (TAA) ions at internal and external sites. Current through single channels was recorded from excised membrane patches of cultured neuroblastoma cells using the patch-clamp technique. All of the TAA derivatives tested blocked the open channel when applied to either side of the membrane. Tetraethylammonium (TEA) reduced the amplitude of current through the open channel. Tetrabutylammonium (TBA) and tetrapentylammonium (TPeA) reduced the open time as a function of the concentration. An additional nonconducting state was observed when TBA or TPeA was applied internally or externally, due to the presence of a drug-bound and blocked state of the channel. The closing rate under control conditions was similar to that in the presence of external tetramethylammonium (TMA), suggesting that channel closing is independent of external drug binding. The concentration for half maximal block of the channel by external TEA was 80 microM. The channel was less sensitive to internal TEA, which half blocked the channel at 27 mM. The dissociation rate of long alkyl chain TAA ions from the channel was slower when applied to the inside, compared to external application, suggesting the presence of distinct internal and external receptors. Long alkyl chain TAA derivatives, such as TBA had a faster association rate with the open channel when applied to the inside of the membrane than when applied to the outside.