T o determine whether ouabain-like compound (OLC) exerts modulatory influences on the activity of vasomotor neurons in the rostral ventrolateral medulla (RVLM), we examined the effects of microinjecting ouabain, digoxin-specific antibody Fab fragments, and mAb against ouabain on the rat RVLM. Microinjection of ouabain into the unilateral RVLM of anesthetized normotensive rats elicited dose-dependent increases in mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA). The pressor and sympathoexcitatory effects of ouabain in the RVLM were reversed by microinjections of an M2 muscarinic antagonist, gallamine, or digoxin-specific antibody Fab fragments. Furthermore, a prior microinjection in the RVLM of gallamine, digoxinspecific antibody Fab fragments, or kainic acid or intravenous injection of hexamethonium all prevented the pressor and sympathoexcitatory effects induced by a subsequent microinjection of ouabain. Microinjections of either digoxinspecific antibody Fab fragments or gallamine per se significantly decreased baseline MAP and RSNA. Injection of digoxin-specific antibody Fab fragments attenuated the effects of a subsequent injection of gallamine. Microinjection of mAb against ouabain, but not nonspecific IgG, also significantly decreased baseline MAP and RSNA. These results suggest that OLC in the RVLM contributes to the tonic activity of vasomotor neurons in anesthetized normotensive rats, and the action of OLC in the RVLM is at least partly mediated by M2 muscarinic mechanisms.