The methylation status of CCGG sites has been determined in long interspersed repeated (L1Hs sequences) DNA and in alphoid satellite DNA extracted from human cell lines and also from pathological specimens. Southern blot experiments were performed using a cloned 1.2 kb KpnI fragment of L1Hs DNA and a cloned 680 bp tetrameric unit of alphoid satellite. DNA as probes for these sequences. In vitro transformation of human lymphocytes by Esptein-Barr virus is correlated with a progressive hypomethylation of L1 Hs DNA sequences. This loss of modification of L1 sequences is also observed in DNA isolated directly from colon adenocarcinoma and in chronic lymphocytic leukemia. Therefore, alteration of the level of methylation of L1 sequences seems not to be due solely to in vitro cultivation of human cells but is associated with the immortalization of these cells. In addition, these sequences are preferentially hypomethylated when compared to alpha-satellite DNA in several lymphoblastoid cell lines and pathological specimens.