The human L1 endonuclease (L1-EN) is encoded by the non-LTR retrotransposon LINE-1 (L1). L1 is responsible for more than 1.5 million retrotransposition events in the history of the human genome, contributing more than a quarter to human genomic DNA (L1 and Alu elements). L1-EN is related to the well-understood human DNA repair endonuclease APE1, and its nicking specificity is a major determinant for retrotransposon integration site selection. The crystal structure of human L1 endonuclease is the first of a retrotransposon-encoded protein and a prototype for retrotransposon-encoded endonucleases involved in target-primed reverse transcription. Structure-based endonuclease alignments reveal a conserved threonine in addition to previously identified invariant residues and suggest that DNA recognition proceeds via the accommodation of an extrahelical nucleotide within a pocket of the enzyme. The present analysis will help to refine phylogenetic and functional relationships among metal-dependent phosphohydrolases and provides a basis for manipulating non-LTR retrotransposon integration site selection.