GABA and enkephalin provide significant innervation of sympathetic preganglionic neurons. Despite some investigation as to the identity of premotor sources of these innervations no comprehensive analyses have been conducted. Similarly, although data describing the cardiovascular effects of blockade of GABAA receptors in the spinal cord is available, the effects at other sympathetic outflows are unknown. In contrast the sympathetic effects of opioid blockade in the spinal cord are unclear. The aims of this study were to identify potential sympathetic premotor sources of GABAergic and enkephalinergic input to the spinal cord and to describe the sympathetic and cardiovascular effects of spinal GABAA receptor and delta/mu opioid receptor blockade in urethane anaesthetised rats. Glutamic acid decarboxylase (GAD67) and preproenkephalin (PPE) mRNA were found in all regions containing sympathetic premotor neurons, with the medullary raphe and RVMM providing the major GABAergic projections, while the PVN, RVMM and medullary raphe provided the major enkephalinergic projections. Intrathecal injection of bicuculline, a GABAA antagonist, elicited large and prolonged increases in all outflows measured, confirming previous work describing a tonic GABAergic influence on vasomotor tone, and revealing a tonic GABAergic inhibition of interscapular brown adipose tissue temperature. Intrathecal naloxone elicited transient small inhibitory effects only on MAP and HR. Thus GABA acting in the spinal cord plays an important role in the tonic suppression of sympathetic outflows while enkephalin appears to play only a minor role.