We measured renal functions and hormones associated with fluid regulation after a bolus injection of aldosterone (Ald) during head-down tilt (HDT) bed rest to test the hypothesis that exposure to simulated microgravity altered renal responsiveness to Ald. Six male rhesus monkeys underwent two experimental conditions (HDT and control, 72 h each) with each condition separated by 9 days of ambulatory activities to produce a crossover counterbalance design. One test condition was continuous exposure to 10° HDT; the second was a control, defined as 16 h per day of 80° head-up tilt and 8 h prone. After 72 h of exposure to either test condition, monkeys were moved to the prone position, and we measured the following parameters for 4 h after injection of 1-mg dose of Ald: urine volume rate (UVR); renal Na⁺/K⁺ excretion ratio; renal clearances of creatinine, Na⁺, osmolality, and free water; and circulating hormones [Ald, renin activity (PRA), vasopressin (AVP), and atrial natriuretic peptide (ANP)]. HDT increased Na⁺ clearance, total renal Na⁺ excretion, urine Na⁺ concentration, and fractional Na⁺ excretion, compared with the control condition, but did not alter plasma concentrations of Ald, PRA, and AVP. Administration of Ald did not alter UVR, creatinine clearance, Ald, PRA, AVP, or ANP but reduced Na⁺ clearance, total renal Na⁺ excretion, urinary Na⁺/K⁺ ratio, and osmotic clearance. Although reductions in Na⁺ clearance and excretion due to Ald were greater during HDT than during control, the differential (i.e., interaction) effect was minimal between experimental conditions. Our data suggest that exposure to microgravity increases renal excretion of Na⁺ by a natriuretic mechanism other than a change in renal responsiveness to Ald.