Intracerebroventricular (i.c.v.) infusion of NaCl mimics the effects of a high-salt diet in salt-sensitive hypertension, raising the sodium concentration in the cerebrospinal fluid (CSF [Na]) and increasing the concentration of an endogenous ouabain-like substance (OLS) in the brain. The OLS in turn inhibits the brain Na, K-ATPase, causing an increase brain renin-angiotensin system (RAS)activity and blood pressure. The Na, K-ATPase (catalytic) isoform(s) that mediate the pressor response to increased CSF [Na] is (are) unknown, but it is likely that one or more isoforms that bind ouabain with high affinity are involved (e.g., the Na, K-ATPase 2 and/or 3 subunits). We hypothesize that OLS-induced inhibition of the 2 subunit mediates this response. Therefore, a chronic reduction in 2 expression via a heterozygous gene knockout (2 +/-) should enhance the pressor response to increased CSF [Na]. i.c.v. infusion of artificial CSF containing 0.225 M NaCl increased mean arterial pressure (MAP) in both +/+ and 2 +/- mice, but to a greater extent in 2 +/-. Likewise, the pressor response to i.c.v. ouabain was enhanced in 2 +/- mice, demonstrating enhanced sensitivity to brain Na, K-ATPase inhibition per se. The pressor response to i.c.v. ANG I but not ANG II was also enhanced in 2 +/- vs. +/+ mice, suggesting an enhanced brain RAS activity that may be mediated by increased brain ACE. The latter hypothesis is supported by enhanced ACE ligand binding in the organum vasculosum laminae terminalis (OVLT). These studies demonstrate that chronic downregulation of Na, K-ATPase 2 isoform expression by heterozygous knockout increases the pressor response to increased CSF [Na] and activates the brain RAS. Since these changes mimic those produced by the endogenous brain OLS, the brain 2 isoform may be a target for the brain OLS during increases in CSF [Na], such as in salt-dependent hypertension.