The effect of probenecid on the metabolic clerance of [35S]furosemide was examined in Sprague-Dawley rats by the single injection technique. Metabolic clearance was found to decrease from 1.74+/-0.11 ml/min in animals which received furosemide alone to 1.06+/-0.10 ml/min in animals which received both furosemide and probenecid. Estimation of urine and biliary clearance revealed that approximately two-thirds of the metabolic clearance was due to urinary clearance and one-third to biliary clearance, and that both decreased after probenecid administration. Thin-layer chromatographic studies indicated furosemide was metabolized by both liver and kidney but also secreted in unchanged form in urine, and that the proportion of unaltered furosemide to metabolite in urine was increased by probenecid. Further studies indicated that phenobarbital pretreatment had no effect on either hepatic or renal clearance of furosemide and that bile clearance was not altered by acute bilateral nephrectomy.