There are no accurate, noninvasive tests to diagnose BK polyomavirus nephropathy, a common infectious complication after renal transplantation. This study evaluated whether the qualitative detection of cast-like, three-dimensional polyomavirus aggregates ("Haufen") in the urine accurately predicts BK polyomavirus nephropathy. Using negative-staining electron microscopy, we sought Haufen in 194 urine samples from 139 control patients and in 143 samples from 21 patients with BK polyomavirus nephropathy. Haufen detection was correlated with pathology in concomitant renal biopsies and BK viruria (decoy cell shedding and viral load assessments by PCR) and BK viremia (viral load assessments by PCR). Haufen originated from renal tubules containing virally lysed cells, and the detection of Haufen in the urine correlated tightly with biopsy confirmed BK polyomavirus nephropathy (concordance rate 99%). A total of 77 of 143 urine samples from 21 of 21 patients with BK polyomavirus nephropathy (disease stages A-C) contained Haufen, and during follow-up (3 to 120 wk), their presence or absence closely mirrored the course of renal disease. All controls were Haufen-negative, however, high viremia or viruria were detected in 8% and 41% of control samples, respectively. kappa statistics showed fair to good agreement of viruria and viremia with BK polyomavirus nephropathy or with Haufen shedding and demonstrated an excellent agreement between Haufen and polyomavirus nephropathy (kappa 0.98). Positive and negative predictive values of Haufen for BK polyomavirus nephropathy were 97% and 100%, respectively. This study shows that shedding of urinary Haufen and not BK viremia and viruria accurately mark BK polyomavirus nephropathy. It suggests that the detection of Haufen may serve as a noninvasive means to diagnose BK polyomavirus nephropathy in the urine.