OBJECTIVE To determine the optimal instruments and techniques for biopsy of upper urinary tract lesions through the small working channel in flexible and small semirigid ureteroscopes. METHODS Urinary tract filling defects and other lesions were accessed with rigid or flexible ureteroscopy and biopsies were done using one or more devices: a 3 F cup biopsy forceps, 2.5 F and 3 F baskets, 2.5 F and 3 F graspers, 3 F snare, brush, and aspiration catheters. All samples, including the cup forceps samples, were sent for cytopathologic study, with those containing grossly visible tissue particles processed as a cell block. Only those specimens reported definitely positive or negative were considered diagnostic, whereas the others were grouped as nondefinitive. Not all sampling techniques could be used in every patient because of the size of the lesion and the technical limitations, including bleeding and instrument position during biopsy. RESULTS There were 55 procedures in 43 patients. The indications included hematuria, filling defect, abnormal cytology, and periodic surveillance. A basket was used in 22 procedures and gave unequivocal results in 15. The other samples were equivocal, nondiagnostic, or unsuitable. The biopsy forceps provided a definitive result in 16 of 21 samples and the grasper was definitive in 5 of 6 samples. We could not obtain a suitable specimen using a snare in 2 cases and the brush gave a definitive result in only 5 of 11 cases. Samples of aspirate and washings were definitive in less than 50% of instances but detected some tumors for which other techniques were equivocal. CONCLUSIONS For the best results, the largest biopsy specimen possible should be obtained. Aspiration or wash alone is often not diagnostic but can give a diagnosis in some patients. Tissue sampling devices, such as the forceps and basket, have an advantage in obtaining a larger sample. Cytopathologic techniques are particularly useful for handling and interpreting the small specimens obtained with ureteroscopic biopsy techniques.