Previous anatomic data have described the distribution of presumptive negative chronotropic and negative dromotropic neurons in the ventro-lateral nucleus ambiguus (NA-VL) following injections of retrograde tracers into physiologically selective parasympathetic intracardiac ganglia. Negative dromotropic neurons were preferentially distributed in the rostral NA-VL (rNA-VL). Negative chronotropic neurons were preferentially distributed in the caudal NA-VL (cNA-VL). Significant numbers of both types of cardio-inhibitory neurons were observed to overlap in an intermediate level of the NA-VL (iNA-VL). In the present report, we have examined the effects of microinjections of the excitatory amino-acid glutamate (GLU) into the cNA-VL and iNA-VL on cardiac rate and AV conduction while recording the electrocardiogram in paced and non-paced cat hearts. The data indicate that: (i) excitation of neurons in the cNA-VL causes a 58 +/- 17% reduction in cardiac rate, without influencing AV conduction; and (ii) excitation of neurons in the iNA-VL causes both a reduction in heart rate (68 +/- 12%) and a decrease in the rate of AV conduction (38 +/- 7%). These physiological results support the anatomical inference that neurons in the cNA-VL that are retrogradely labeled from physiologically selective parasympathetic intracardiac ganglia selectively exhibit negative chronotropic properties. Furthermore, the data indicate that there is a longitudinal cardiotopic organization of both negative chronotropic and negative dromotropic neurons in the NA-VL. This CNS organization mirrors the peripheral organization of functionally selective cardiac components of the vagus nerve. Finally, the data are consistent with the hypothesis that anatomically separated and functionally selective parasympathetic preganglionic vagal motoneurons in the NA independently control cardiac rate and AV conduction.