The renal clearance of endogenous creatinine is widely used to assess glomerular filtration rate (GFR) and renal function in animal investigations. The objective of the present investigation was to evaluate the extent of renal secretion of endogenous creatinine in rats and the effect of probenecid, the classical inhibitor of organic anion transport, on creatinine clearance. Ten female Lewis rats received 3H-inulin (5-muCi i.v. bolus followed by 5 muCi/hr) throughout a 6-hr period. Three hours after initiation of the inulin infusion, probenecid was administered (92.4-mg/kg i.v. bolus followed by 0.59 mg/min/kg). Steady-state serum concentrations of about 500 micrograms/ml probenecid were achieved. Renal clearance was assessed between 1 and 3 hr (control) and between 4 and 6 hr (probenecid treatment). A preliminary study in seven rats demonstrated no time-dependent change in inulin or creatinine clearance between these two study intervals. Creatinine clearances were determined by an alkaline picrate assay which incorporated Fuller's earth (Lloyd reagent) to remove interfering noncreatinine chromogens from serum samples and these values were compared with those using a nonspecific picrate assay. "True" clearance ratios of creatinine to inulin (Clcr/CLin) were greater than unity (2.33 +/- 0.83, mean +/- SD) and were significantly decreased after probenecid treatment (1.26 +/- 0.28, P less than 0.01). Probenecid had no effect on GFR, as assessed by inulin clearance. Using the nonspecific picrate assay, CLcr/CLin was 1.12 +/- 0.41, which was not significantly different from unity and which decreased to 0.53 +/- 0.12 after probenecid treatment. Therefore, creatinine undergoes extensive renal secretion in female Lewis rats.