The brain atrial natriuretic peptide (ANP) neuronal system appears to be involved in the increase in plasma ANP which follows blood volume expansion in the rat. To determine if this neuronal system is essential to the natriuresis and increase in plasma ANP which follow volume expansion, highly specific antiserum against ANP (ANP-AB) and/or normal rabbit serum as a control was microinjected into the third cerebral ventricle (3V) of conscious rats, and the effect on the natriuresis and increase in plasma ANP induced by intravenous injection of 2 ml/100 g body weight of 0.3 M NaCl was examined. Although there was no effect of ANP-AB on initial levels of plasma ANP or natriuresis 3 h after 3V injection, the natriuresis in response to blood volume expansion was significantly inhibited. The increase in plasma ANP which followed volume expansion was also significantly reduced at 5 min but recovered at 15 min. The results indicate that the brain ANP neuronal system plays an essential role in the mediation of volume expansion-induced increase in plasma ANP and natriuresis. The failure to block these responses completely may be due to the use of an inadequate dose of antiserum or other brain mechanisms may be able to mediate these responses.