Replication of the human polyomavirus BK (BKV) in renal tubular epithelial cells causes viruria and BKV-nephropathy in kidney transplant recipients. Following prolonged high-level BKV replication, rearrangement of the archetype non-coding control region (NCCR) leads to a mixture of BKV variants. The aim of this study was to compare potential functional differences of 12 rearranged (rr)-NCCR variants with the archetype (ww)-NCCR (WWT) found in allograft biopsies or urine from three kidney transplant recipients including two with BKV-nephropathy. Twelve different rr-NCCRs and one archetype ww-NCCR were inserted between the early and late protein coding region of BKV(Dunlop) to make recombinant BKV genomes for transfection into Vero cells. Immunoblotting, immunofluorescence staining, and quantitative PCR demonstrated that viral protein expression and extracellular BKV loads of 10 rr-NCCR variants were similar or higher than observed for the ww-NCCR BKV. Two rr-NCCR variants (RH-2 and RH-19) were non-functional. The functional rr-NCCRs produced infectious progeny successfully infecting primary renal proximal tubular epithelial cells. The number of infected cells and extracellular BKV loads corresponded to the activity seen in Vero cells. Three rr-NCCR variants (RH-1, RH-10, RH-13) only gave rise to a few infected cells similar to ww-NCCR, whereas seven variants had intermediate activity (RH-5, RH-6, RH-8, RH-9, RH-11) or high replication activity (RH-7 and RH-18) with several hundred infected cells per well. The results indicate that both functional and non-functional BKV rr-NCCR variants arise during BKV replication in kidney transplant recipients and that most functional rr-NCCR variants confer a higher replication capacity than archetype ww-NCCR. J. Med. Virol. 81:1959–1967, 2009. © 2009 Wiley-Liss, Inc.