In a previous analysis on the rabbit S-A node the ACh-induced current was separated from the membrane current by subtracting the control from the current recorded in presence of ACh. In view of a possible interference of K accumulation processes, in the present paper the validity of the subtraction method was tested by studying the direct and indirect effects of ACh on the outward potassium current (iK). The following results were obtained. (1) The ACh-dependent channel activation and the iK-channel activation are different processes. (2) The activation curve of iK and the time constant of decay of iK current on return from a depolarizing clamp pulse were not affected by ACh. (3) In the majority of the experiments the presence of an accumulation component in the extra-current elicited by ACh could not be resolved. In a few cases the amplitude of the tail current was decreased in the presence of ACh. (4) In the case where iK was reduced, the fully-activated current-voltage relationship (i/K) was altered in the same way as that observed when the external K concentration was increased. In this case the difference between the control and the current recorded in the presence of ACh yielded a current component having a time constant similar to that of iK. We concluded that the decrease in the amplitude was due to an increase in K concentration in the clefts between the cells (K accumulation), associated with ACh application. No direct effect of ACh on the iK channel is apparent. (5) Because of the difference in the time constants of the relaxation current and the current change induced by accumulation the two processes could be clearly separated from each other.