Polyomavirus BK (BKV) causes asymptomatic latent infection in the human host that is reactivated during periods of immune suppression. Detection by conventional tube cell culture is difficult and time consuming because BKV exhibits slow growth with late (14 to 28 days) and subtle cytopathic effects. We developed a shell vial cell culture assay (SVA) using a cross-reactive monoclonal antibody to the T antigen of simian virus 40 to detect BKV rapidly by indirect immunofluorescence. Nuclear fluorescence was seen in BKV-infected cells as early as 16 h postinoculation; 6 to 28 times more foci were present at 36 h postinoculation. Human embryonic kidney cells infected with BKV produced 7 to 42 times more fluorescent foci than MRC-5 or rhabdomyosarcoma cells did. Centrifugation enhanced the infectivity of BKV in the SVA. To define the clinical utility of SVA, urine specimens from organ transplant patients were tested. Of 27 patients, 4 (15%) were found to be positive by SVA. SVA offers a simple and rapid method for detection of BKV that can be of use in clinical studies of this virus.