We investigated whether low arterial oxygen tension (PaO₂) or hypoxia-induced plasma volume (PV) contraction, which reduces central blood volume (BV) and atrial distension, explain reduction in circulating atrial natriuretic peptide (ANP) after prolonged hypoxic exposure. Ten healthy males were exposed for 4 days to hypobaric hypoxia corresponding to an altitude of 3,500 m. PV changes were determined by carbon monoxide rebreathing. Venous plasma concentrations of midregional proANP (MR-proANP) were measured before and at the end of the exposure. At the latter time point, the measurement was repeated after 1) restoration of PaO₂ by breathing a hyperoxic gas mixture for 30 min and 2) restoration of BV by fluid infusion. Correspondingly, left ventricular end-diastolic volume (LVEDV), left atrial area (LAA), and right atrial area (RAA) were determined by ultrasound before exposure and both before and after fluid infusion at the end of the exposure. Hypoxic exposure reduced MR-proANP from 37.9 ± 18.5 to 24.5 ± 10.3 pmol/L ( P = 0.034), LVEDV from 107.4 ± 33.5 to 91.6 ± 26.3 mL ( P = 0.005), LAA from 15.8 ± 4.9 to 13.3 ± 4.2 cm² ( P = 0.007), and RAA from 16.2 ± 3.1 to 14.3 ± 3.5 cm² ( P = 0.001). Hyperoxic breathing did not affect MR-proANP (24.8 ± 12.3 pmol/L, P = 0.890). Conversely, fluid infusion restored LVEDV, LAA, and RAA to near-baseline values (108.0 ± 29.3 mL, 17.2 ± 5.7 cm² , and 17.2 ± 3.1 cm² , respectively, P > 0.05 vs. baseline) and increased MR-proANP to 29.5 ± 13.3 pmol/L ( P = 0.010 vs. preinfusion and P = 0.182 vs. baseline). These findings support that ANP reduction in hypoxia is at least partially attributed to plasma volume contraction, whereas reduced PaO₂ does not seem to contribute.