This study assesses the contribution of metabolism for the disposition of pentamidine in the rat. With the use of 14C-labelled compound, the excretion of radioactivity in urine and faeces has been studied in four rats during 44 days after a single intravenous injection of the drug. The urinary and faecal excretion of the radioactivity were of equal importance; 22 +/- 2% (mean +/- S.D.) and 25 +/- 4% being detected in urine and faeces, respectively. The activity in organs and tissues at 44 days after drug administration was also measured and amounted to 21 +/- 5% of the administered dose. Using HPLC the proportion of metabolites in urine in relation to unchanged pentamidine increased with time after dose, being 76 +/- 15% (mean +/- S.D.) of the total excreted radioactivity on day 1 and 97 +/- 1% on day 6. HPLC--tandem mass spectometry was used for identification of metabolites in urine obtained from four rats given unlabelled pentamidine. Using synthetic reference compounds and the selective MS/MS mode four oxidized metabolites of pentamidine were identified either by direct injection into the system or by analyses of extracted urine. Thus, a substantial part of pentamidine is excreted as metabolites in urine.