Purity of different batches of [3H]inulin delivered from leading manufacturers was elevated with a chromatographic method (Sephadex G-25 column) that allowed simultaneous analysis of cold inulin, [3H]inulin, and [14C]inulin in the same run. Among four batches of [3H]inulin received within 5 mo, two were found relatively pure, whereas two were partly decomposed to lower-molecular-weight fragments. The chromatographic profile of pure isotopes was not significantly affected by redistribution and freeze drying, nor by subsequent storage in the freeze-dried state at -20 degrees C for up to 5 mo, nor by incubation in aqueous solution at 37 degrees C for 24 h. Three batches of [3H]inulin with different grades of decomposition (noninulin percentages 13%, 38%, and 61%, respectively) were selected for clearance experiments and infused simultaneously with cold and undecomposed [14C]inulin to conscious rats. [14C]inulin had a significantly higher clearance than cold inulin (+7.6 +/- 0.6%) and relatively pure [3H]inulin (+12.4 +/- 0.4%). Decomposed [3H]inulin isotopes progressively underestimated clearance of cold inulin to an extent related to the degree of decomposition. Thus at the end of the 5-h clearance experiment, ratios between clearance of tracer and of cold inulin were 0.92, 0.71, and 0.60 for the three 3H isotopes, respectively. This study indicates that [3H]inulin delivered from leading manufacturers may be decomposed to an extent that invalidates its use as a marker for glomerular filtration rate (GFR). It is thus necessary to check the purity routinely before use. Within the same rat, clearance of undecomposed [3H]inulin and [14C]inulin may differ by 12%, and for this reason they should not be used interchangeably as GFR markers.