Long interspersed element-1 (LINE-1) retrotransposons from Homo sapiens (L1Hs) are not expressed in normal somatic cells. In malignant cells, there is a direct correlation between the hypomethylation of 5' L1Hs sequences and the presence of L1Hs proteins, suggesting that elements with hypomethylated 5' ends are transcriptionally active. Sequences flanking the 5' ends of hypomethylated L1Hs elements were isolated from the T-47D breast cancer cell line by inverse-PCR and cloning. These flanker clones served as probes for analyzing the loci harboring the hypomethylated L1Hs elements. Sequencing demonstrated that the flankers have no homology with one another and do not appear to contain common short motifs that might serve as recognition sites for regulatory factors. The hypomethylated L1Hs elements are located on many chromosomes, although three of twelve are on chromosome 15. Southern blotting indicates that certain elements are hypomethylated in numerous cell lines, and that elements that are hypomethylated in malignant germ cells comprise a different subset of elements than those that are hypomethylated in non-germ cell malignancies. These results suggest that the subset of L1Hs elements that is hypomethylated in malignant cells is not simply a random collection of L1Hs elements.