We have developed a method of analyzing individual cells to detect proviral DNA of the bovine leukemia virus (BLV) using flow cytometry and PCR. Individual cells of the BL3* cell line, which contain multiple integrated copies of the BLV provirus, and the uninfected cell line BL3°, were sorted into wells of a 96-well plate. Following cell lysis, portions of the BLV envelope (ENV) and cellular prolactin (PRL) genes were amplified simultaneously using PCR. Viral and cellular products of first-round PCR were amplified separately in a second round of PCR using "heminested" primers. Separation of the PCR products by polyacrylamide gel electrophoresis yielded distinct fragments of the predicted sizes. The operational sensitivity of this method for the detection of virus was >90% when testing single infected cells. In addition, we were able to reliably amplify DNA from a single BL3* cell among as many as 105 BL3° cells and established that the sensitivity for detecting a single infected cell among 20, 100, or 1000 uninfected cells was at least 90%. Estimates of low percentages of infected cells were obtained by applying probability theory to results of experiments conducted on wells containing more than one cell. Using these methods, B lymphocytes obtained from the peripheral blood of BLV-infected cattle were tested for proviral DNA. BLV ENV was identified in 76.9 ± 4.9% of single B cells tested from a seropositive animal with persistent lymphocytosis (PL), but in only 0.033 ± 0.009% of B cells from another seropositive cow without PL. This method of analyzing individual or small num-bers of cells for the presence of specific DNA sequences provides a sensitive and quantitative tool for the study of the pathogenesis of infectious, neoplastic, and hereditary diseases at the single-cell level.