Protocols were designed to 1) identify atrial natriuretic factor (ANF) bioactivity in pregnant rats, 2) compare ANF activity in atria from gravid and virgin Sprague-Dawley animals, and 3) assess renal and vascular responsiveness to ANF during gestation. Similar quantities of ANF were extracted from atria of term gravid and age-matched controls (assayed as the natriuretic response in awake virgins). We then purified by high-performance liquid chromatography a peptide identical to the synthetic 23 amino acid atriopeptin II (AP II). With equimolar injections of ANF, gravid rats had a blunted natriuretic response (P less than 0.05). However, when dose was based on estimated extracellular volume, awake virgin and pregnant animals displayed similar highly significant dose-response curves to infused AP II (R's 0.8). When hydropenic rats were assayed, both gravid and virgins required 10 times more ANF to initiate a diuresis; dose-response curves again being similar and highly significant. Decrements in blood pressure were also similar in pregnant and nongravid animals. Thus, despite an increased intravascular volume of 70% during gestation, cardiac content of ANF and the renal and vascular responsiveness to this peptide are similar to those in virgin rats, suggesting resetting of ANF-volume relationships during pregnancy.