Central actions of estrogen (E2) include, among others, modulation of autonomic and cardiovascular function. Despite the well-known influence of sex steroid hormones on the incidence of cardiovascular disorders, little is known about the neural substrates and receptors mediating central E2 actions on autonomic function. The paraventricular nucleus of the hypothalamus (PVN) is an important site for the integration of neuroendocrine and autonomic function. Interestingly, while this region was originally found to lack the classical ERalpha receptor, recent studies demonstrated a high degree of expression of the ERbeta subtype. To determine specifically whether autonomic-related neurons in the PVN express ERbeta, thus constituting a neuronal substrate for central E2 actions on autonomic function, we carried out an immunohistochemical study of ERbeta expression in a subpopulation of PVN neurons that innervate the rostroventrolateral medulla (RVLM). ERbeta immunostained neurons were found in medial and caudal aspects of the PVN, overlapping with the distribution of RVLM-projecting neurons. Overall, approximately 50% of RVLM-projecting PVN neurons expressed ERbeta immunoreactivity. Interestingly, the degree of colocalization was found to be sex-dependent (higher expression in males), and varied according to the topographical distribution of neurons within the PVN. ERbeta immunoreactivity was also observed in magnocellular compartments of the PVN, although this appeared to be consistently weaker than that observed in autonomic-related subnuclei. These studies demonstrate for the first time ERbeta expression in identified autonomic-related neurons in the PVN, and suggest that these neurons constitute an important neuronal substrate mediating E2 actions on autonomic and cardiovascular control.