We recently purified and cloned the gene for a DNA structure-specific endonuclease, FEN-1, from murine cells. The murine protein recognizes 5' DNA flap structures that have been proposed in DNA replication, repair, and recombination. Here, we report the sequence of the human FEN1 gene. The translated sequence is identical to peptide sequence obtained from maturation factor-1, which is 1 of the 10 essential proteins for cell-free DNA replication. The human protein has the same structure-specific DNA endonuclease activity as the murine protein. Two human chromosomal hybridization signals, 11q12 and 1p22.2, were observed by FISH analysis using human genomic clones homologous to the mouse Fen-1 gene. The localization on human 11q12 was confirmed using radiation-reduced hybrids. The mouse Fen-1 gene is assigned to chromosome 19 based on somatic cell hybrids. The significance of these FEN1 gene localizations in human and mouse is discussed.