To assess the contribution of putative neurotransmitters in mediating changes in adrenal and autonomic function evoked by activation of medullary dorsal horn neurons, microinjections of substance P, bicuculline methiodide, or muscimol were directed at various laminae of trigeminal subnucleus caudalis in the anesthetized cat. Injections of substance P (35.6 pmol) into the superficial layers (lamina I-II) of subnucleus caudalis increased the adrenal secretion of epinephrine (+8.3 ± 2.3ng/min, P < 0.01), arterial pressure (+11 ± 5.3mm Hg, P < 0.01), and heart rate (+19.4 ± 4.9beats/min, P < 0.01) by 1 min, and increased the plasma concentration of adrenocorticotropin (+26 ± 10pg/ml, P < 0.01) by 3 min. Substance P injections into the magnocellular layers (lamina III-IV) or deep magnocellular layers (lamina V-VI) had no significant effects. Microinjections of the GABA A antagonist, bicuculline methiodide (62.4 pmol), into the superficial layers of subnucleus caudalis increased the adrenal secretion of epinephrine (+4.5 ± 3.2ng/min, P < 0.01) by 1 min, whereas injections of the GABA A agonist, muscimol (280 pmol), decreased the secretion (−5.8 ± 2.8ng/min, P < 0.05) by 6 min. Arterial pressure increased after bicuculline (+17.8 ± 8.2mm Hg, P < 0.01) and decreased after muscimol (−6.3 ± 2.9mm Hg, P < 0.01) injections into the superficial layers. Injections of bicuculline or muscimol into the magnocellular layers or into the deep magnocellular layers had no effect on adrenal secretion of catecholamines or on systemic cardiovascular function. Peripheral venous concentrations of adrenocorticotropin were not affected significantly by microinjections of GABAergic agents regardless of the laminar site of injection within subnucleus caudalis. Equivalent volume injections of artificial cerebrospinal fluid into the superficial laminae of subnucleus caudalis had no significant influence on any measured variable. Substance P-evoked changes in the adrenal secretion of epinephrine were not correlated with changes in adrenal venous blood flow, whereas bicuculline- and muscimol-evoked changes in adrenal secretion of catecholamines were positively correlated with changes in adrenal blood flow ( P < 0.01). The results indicate that substance P and GABA contribute significantly to the trigeminal control of adrenal and autonomic function by acting on neurons in the superficial layers of subnucleus caudalis, a brainstem region that processes nociceptive sensory information.