The acute effect of i.v. spironolactone dissolved in ethanol on renal digoxin clearance was studied in 10 dogs. All dogs received i.v. digoxin (0.02 micrograms/kg/min) and 0.9% saline (30 ml/kg of bolus and 0.1 ml/kg/min for maintenance) throughout the study. After 2 hr of equilibration, four 15-min urine collections were made. The dogs then received, over 1 hr, a loading infusion of 4 mg/kg of spironolactone in ethanol (group 1, N = 6) or ethanol alone (group 2, N = 4). Thereafter the infusion was maintained at one-tenth the initial rate for eight 15-min periods. Three additional control dogs received ethanol plus spironolactone without digoxin. Digoxin radioimmunoassay of plasma and urine from these control animals did not reveal any significant cross-reactivity between spironolactone and the antibody of the digoxin radioimmunoassay. The digoxin clearance (CDIG) and ratio of CDIG to inulin clearance (CDIG/CIN) increased insignificantly with ethanol alone, but were respectively reduced by 21 and 28% with spironolactone (P less than .05). The reductions of CDIG and CDIG/CIN were observed immediately after the spironolactone loading infusion and were associated with a sudden rise in plasma digoxin. The changes in digoxin handling with spironolactone appeared to be temporally dissociated from the antimineralocorticoid activity of spironolactone. We conclude that spironolactone inhibits renal CDIG by a mechanism that may be independent of its antimineralocorticoid activity.