Several human DNAs digested with Kpn I restriction endonuclease released a 0.6-kilobase (kb) segment that varied in its intensity among human samples. A recombinant DNA clone (N6.4) of these 0.6-kb Kpn I segments was isolated and used to probe the genomic content and restriction cleavage pattern of homologous sequences. The hybridization patterns revealed a previously undescribed, moderately repetitive long interspersed (LINE) sequence family, which we have termed L2Hs (second LINE family in Homo sapiens). This LINE family exhibits both quantitative and qualitative polymorphisms in the human population. The content of L2Hs sequences in human genomes varies over a 5-fold range. Relative to the value for a human placental DNA, sequences homologous to the L2Hs family occur in lower amounts in gorilla DNA (approximately 20%) and even less in DNA from chimpanzees and other primates (less than 1%). Thus, the L2Hs sequences appear to have emerged only recently as a moderately repetitive sequence family in primate evolution. The observed restriction fragment length polymorphism of the L2Hs family members may reflect patterns of sequence rearrangements, amplifications, and/or deletions in human genomes.