This study was performed to help elucidate the role of spinal cholinergic neurons in cardiorespiratory function by selective activation of spinal or medullary cholinergic systems in anesthetized rats. A selective site of action of cholinergic drugs on the spinal cord was obtained by refining the method of intrathecal (i.t.) drug injection to localize drug distribution to specific spinal segments. I.t. injection of the cholinesterase (ChE) inhibitor, neostigmine (NEO), produced a significant reduction in spinal, but not medullary tissue levels of ChE, and evoked marked pressor and tachycardic responses without any changes in respiratory parameters. In contrast to i.t. injection, intracisternal (i.c.) injection of NEO which inhibited both spinal and medullary ChE, produced characteristic respiratory changes — increased tidal volume and decreased respiratory rate and minute volume, as well as pressor and tachycardic responses. I.t. injection of the muscarinic antagonist, methylatropine, inhibited the cardiovascular responses to i.t. NEO, but not the cardiorespiratory responses to i.c. NEO. These cardiovascular responses to i.t. NEO were blocked by spinal transection, but not by midcollicular transection. Finally, the pressor and tachycardic responses to i.t. NEO were inhibited following peripheral α-adrenergic and β-adrenergic blockade, respectively. These results indicate that activation of the spinal cholinergic system selectively produces a sympathoexcitatory response through spinal muscarinic receptor activation independent of respiratory changes. This finding is consistent with the possibility that such responses are elicited by activation of a non-cholinergic bulbo-spinal sympathoexcitatory pathway at the spinal level, or at higher centers through an ascending pathway. In either case, the spinal cholinergic system appears to be anatomically and pharmacologically distinct from the medullary pathway and may subserve a different function.