To investigate the hypothesis whether the hypothalamus releases an active (ouabain-sensitive) sodium transport inhibitor, we cultured hypothalamic and cortical cells from day 17 fetal rats. Culture media from hypothalamic cells reduced the total erythrocyte sodium efflux rate constant from 0.487 +/- (SE) 0.014 to 0.408 +/- 0.013 (P less than 0.001), and the ouabain-sensitive rate constant from 0.305 +/- 0.015 to 0.240 +/- 0.016 (P less than 0.01). Hypothalamic media also showed a dose-dependent displacement of [3H]-ouabain-binding to erythrocyte membranes. Neither cortical nor conditioned media (incubated without cells) had any effect. Various well-characterized hormones of hypothalamic origin failed to inhibit sodium efflux rate constant. These studies demonstrate that fetal rat hypothalamic cells contain and release a factor which inhibits sodium transport in human erythrocytes.