The effects of pentobarbital, phenobarbital, amobarbital, and diphenylbarbituric acid were studied by examining the average lifetime and conductance of acetylcholine-activated sodium channels in Aplysia neurons. Although none of the barbiturates tested modified the conductance of single-ion channels, pentobarbital and amobarbital had profound effects on channel lifetime. In the absence of barbiturate, relaxations in response to voltage jumps during steady-state current responses to acetylcholine have a single-exponential time course. In the presence of pentobarbital (75-500 microM), current relaxations consist of two exponential components that take the same direction as control relaxations. The faster component becomes faster and relatively larger at higher pentobarbital concentrations, while the slower component always has the same time constant as control. These results are not consistent with a sequential model of channel blockade described for local anesthetics, in which blocked channels must become unblocked before channel closure can occur. Kinetic data are better explained by a cyclic model in which blocked channels have the same probability of closing as nonblocked channels. Alternatively, the results can also be explained by a two-site model in which one binding site regulates the susceptibility of channels to the effects of the barbiturate, whereas occupation of the second site determines the extent of changes in channel lifetime. The effects of amobarbital were qualitatively similar to those of pentobarbital, while phenobarbital and diphenylbarbituric acid did not alter current relaxations at similar concentrations.