Elevated plasma concentrations of aldosterone (ALDO) are observed in patients treated with spironolactone. Because ALDO is eliminated via UGT2B7-catalyzed 18β-glucuronidation, this study aimed to determine whether spironolactone and its primary metabolites, canrenone and canrenoic acid, inhibit ALDO 18β-glucuronidation by recombinant UGT2B7 and by human liver (HLM) and human kidney cortical (HKCM) microsomes. Initial experiments characterized the effects of all three compounds on 4-methylumbelliferone and ALDO glucuronidation by recombinant human UGT2B7. IC50 values for spironolactone and canrenone ranged from 26 to 50 μM, whereas canrenoic acid was a weak inhibitor. Inhibitor constant (Ki) values for spironolactone and canrenone inhibition of ALDO 18β-glucuronidation were subsequently determined with HLM, HKCM, and UGT2B7 as the enzyme sources. Spironolactone and canrenone were competitive inhibitors of ALDO 18β-glucuronidation by HLM, HKCM, and UGT2B7. Mean (±) Ki values for spironolactone were 52 ± 22 (HLM) and 34 ± 4 μM (HKCM), and mean (±) Ki values for canrenone were 41 ± 19 (HLM) and 23 ± 2 μM (HKCM). Ki values for spironolactone and canrenone inhibition of ALDO 18β-glucuronidation by recombinant UGT2B7 were 23 and 11 μM, respectively. "Actual" Ki values for spironolactone and canrenone inhibition of ALDO 18β-glucuronidation, which take into account the role of endogenous microsomal inhibitors, are predicted to be 3 to 5 and 2 to 4 μM, respectively. The data indicate that the elevated ALDO concentrations observed in patients treated with spironolactone may be due, at least in part, to a pharmacokinetic interaction, and spironolactone and canrenone should be considered to be potential inhibitors of the UGT2B7-mediated metabolic clearance of drugs in both liver and kidney.