In this study, we validated a method for repetitive measurements of the glomerular filtration rate (GFR) in conscious rats. We applied this method to estimate GFR before and after induction of diabetes in the same rats. Furthermore, acute administration of C-peptide reduces GFR only in diabetic rats without affecting renal blood flow. We therefore also determined the GFR response to C-peptide administration during both glycemic states. GFR was estimated from plasma clearance curves using a single bolus injection of freshly dialyzed FITC-inulin, followed by serial blood sampling over 155 min. FITC-inulin clearance was calculated using non-compartmental pharmacokinetic data analysis. Baseline total GFR in normoglycemic control rats was 2.10±0.18 ml/min, and was unaffected by C-peptide (2.23±0.14 ml/min). Diabetic rats had elevated GFR (3.06±0.34 ml/min), which was normalized by acute C-peptide administration (2.35±0.30 ml/min). In conclusion, this method for estimation of GFR in conscious animals result in values that are in good agreement with those obtained from traditional GFR measurements on anaesthetized rats. However, multiple measurements from the same conscious subjects can be obtained using this method with the additional advantage of avoiding radioactive contamination of cages and bedding.