The potassium channel blocker 4-aminopyridine (4-AP) has been shown to antagonize the negative inotropic effects of muscarinic receptor agonists on atrial preparations. This is consistent with muscarinic agonists mediating their negative inotropy through the opening of potassium channels. In the present study, the possibility of a direct antagonism of the muscarinic receptor was examined by comparing the effects of 4-AP upon the responses to carbachol of isolated left atria (negative inotropy) and tracheal spirals (contraction) from reserpine pretreated guinea-pigs. The latter response is K+ channel-independent. The concentration-response curve for carbachol on the paced left atria was displaced 520-fold to the right by 4-AP (10 mM). 4-AP alone caused dose-related contractions of the tracheal spirals. Carbachol-induced contractions were, however, superimposed upon the raised tone and there were substantial rightwards shifts of the concentration-response curves of 4.7- and 31.4-fold by 1 and 10 mM of 4-AP, respectively. Thus 4-AP appears to have muscarinic receptor antagonistic blocking properties. The blockade of the atrial responses was, however, substantially greater and could be attributed to an additional blockade of muscarinic receptor-linked potassium channels. The negative inotropic responses of the A1-adenosine receptor agonist L-phenylisopropyladenosine (L-PIA) were also antagonized by 4-AP, but to a lesser extent than for carbachol. After allowing for the muscarinic receptor blocking activity of 4-AP, carbachol was still antagonized more effectively than L-PIA. This could be due to the presence of a K+ channel-independent component in the response to L-PIA which is not susceptible to 4-AP.