The effects of 4-aminopyridine (4-AP, 0.3 to 300 micrograms) on atrial rate and atrial muscle contractile force were investigated in the isolated, blood-perfused dog atrial preparation. Low doses (less than 30 micrograms) of 4-AP injected into the sinus node artery caused a dose-dependent positive inotropic response and a negligible positive chronotropic response. A higher dose (300 micrograms) of 4-AP induced a transient positive chronotropic response followed by a negative response and positive and negative inotropic responses. The negative cardiac responses to 4-AP were completely inhibited by an adequate dose of atropine and were potentiated by physostigmine. TTX or C6 suppressed partly, but significantly the negative responses induced by 4-AP. On the other hand, the negative responses to electrical stimulation (ES) were inhibited by atropine, C6 and TTX and those to ACh were blocked by atropine but not by C6 or TTX. The positive responses to 4-AP were not affected by propranolol but were potentiated by atropine. The positive inotropic response to 4-AP was significantly suppressed by a calcium channel blocker, diltiazem. From these results, it is concluded that the cardiac responses to 4-AP are composed of direct positive responses and negative responses which are related to activation of parasympathetic ganglionic neurotransmission.