To investigate interactions between sites in the medulla which control the sympathetic (SNS) and the parasympathetic (PNS) nervous system, the activity of cardiovascular neurons in the rostral ventrolateral medulla (RVLM) was recorded during chemical stimulation of cardioinhibitory neurons in the nucleus ambiguus (NA). Cardioinhibitory sites in the NA were identified in urethane anesthetized rats. The spontaneous activity of single units in the RVLM was recorded and cardiovascular units were identified as units which displayed a cardiac cycle-related rhythmicity (CR) and decreased activity after i.v. phenylephrine. Of 55 units studied, 36 units were identified as cardiovascular and of these, 24 decreased activity after glutamate (GLU) microinjection in the NA, two units were excited and ten did not respond. Decreases in RVLM unit activity were positively correlated with the magnitude and duration of heart rate decreases elicited by GLU. Seven of the 24 units were tested for their response to microinjection of glycine (GLY) in the NA. GLY did not change firing frequency but did accentuate CR. These results demonstrate an inhibitory connection between the NA and RVLM which influences the rhythmicity of RVLM unit activity and provide clear evidence for functional interactions between the SNS and PNS within the central nervous system.