Vagal stimulation results in complex changes of pacemaker excitability in the sinoatrial node (SAN). To investigate the vagal effects in the rabbit SAN, we used optical mapping, which is the only technology that allows resolving simultaneous changes in the activation pattern and action potentials morphologies. With the use of immunolabeling, we identified the SAN as a neurofilament 160-positive but connexin 43-negative region (n = 5). Normal excitation originated in the SAN center with a cycle length (CL) of 405 +/- 14 ms (n = 14), spread anisotropically along the crista terminalis (CT), and failed to conduct toward the septum. Postganglionic nerve stimulation (PNS, 400-800 ms) reduced CL by 74 +/- 7% transiently and shifted the leading pacemaker inferiorly (78%) or superiorly (22%) from the SAN center by 2-10 mm. In the intercaval region between the SAN center and the septal block zone, PNS produced an 8 +/- 1-mm(2) region of transient hyperpolarization and inexcitability. The first spontaneous or paced excitation following PNS could not enter this region for 500-1,500 ms. Immunolabeling revealed that the PNS-induced inexcitable region is located between the SAN center and the block zone and has a 2.5-fold higher density of choline acetyltransferase than CT but is threefold lower than the SAN center. The fact that the inexcitability region does not coincide with the most innervated area indicates that the properties of the myocytes themselves, as well as intercellular coupling, must play a role in the inexcitability induction. Optically mapping revealed that PNS resulted in transient loss of pacemaker cell excitability and unidirectional entrance conduction block in the periphery of SAN.