As recent immunohistochemical evidence has shown the coexistence of putative neurotransmitters in the rostral ventrolateral medulla (RVLM), we have investigated the possibility that there may be an interaction of putative transmitters on the firing frequency of cardiovascular neurons in the RVLM. Extracellular activity was recorded from 37 spontaneously firing units in the right RVLM of urethane anaesthetized and artificially ventilated rats. Nine of these units were classified as cardiovascular neurons because: (i) they were silenced by baroreceptor activation (1-3-mu-g phenylephrine i.v.); and (ii) they showed rhythmicity of their spontaneous activity in synchrony with the cardiac cycle. Microiontophoresis of combinations of near threshold amounts of L-glutamate (GLU; 10 nA), acetylcholine (Ach; 30 nA) and substance-P (SP; 60 nA) showed a synergistic interaction of these substances with one another in eliciting changes in firing frequency of cardiovascular neurons. These results show that GLU and Ach, GLU and SP and Ach and SP interact synergistically to influence the firing frequency of cardiovascular neurons in the RVLM and suggest that these substances play a physiological role in the neural control of the circulation.