In order to examine the role of Epstein-Barr virus (EBV) in the immortalization of human B lymphocytes and in the pathogenesis of lymphoid malignancies, we investigated whether the EBV integration into the human genome is randomly distributed or whether the virus integrates preferentially at certain sites. Twelve in vitro immortalized human lymphoblastoid cell lines (LCLs), two in vivo infected LCLs, and one Burkitt's lymphoma cell line (EB2) were examined by non-radioactive in situ hybridization (ISH) with a biotinylated EBV probe. Recurrent hybridization sites were detected in all 15 cell lines. The chromosomes frequently carrying the EBV genome were chromosomes 1, 2, 4, and 5. In more than 70 chromosomal bands, a greater number of integration sites than expected was found (p < 0.05). Approximately half of these bands were involved in the majority of the cell lines (for example, 1p31, 1q43, 2p22, 3q28, 4q13, 5p14, 5q12, and 11p15) whereby band 5p14 was involved in all LCLs analyzed. Virtually no viral integrations were found on the sex chromosomes (X, Y). The majority of the EBV integrations was found in G-band-positive material (p < 0.0001). Thus, our findings clearly show that EBV integrates into the human genome in a non-random manner.