Flap endo/exonuclease-1 (FEN-1) recognizes 5'-flap DNA structures that have been proposed to be important intermediates in DNA replication, repair and recombination, and cleaves the double strand-single strand junction of flap substrates. Using an in vitro model system, recent studies have shown that FEN-1 is a necessary enzyme for the removal of RNA primers in Okazaki fragment maturation during lagging strand DNA synthesis. In this report, the FEN-1 gene expression was examined during cell cycle and differentiation. Although FEN-1 mRNA and protein could be detected at all stages of the cell cycle, their levels were more elevated in exponentially proliferating cells than in G1 or G2/M-synchronized cells. Moreover, a significant increase of FEN-1 protein was observed when temporarily quiescent fibroblasts were induced to proliferate by serum stimulation. In contrast, the FEN-1 mRNA level showed a sharp decrease in HL-60 cells differentiated by dimethyl-sulfoxide, all-trans retinoic acid or 12-O-tetradecanoylphorbol-13-acetate. These results demonstrate that the FEN-1 gene expression is up-regulated during entrance into the mitotic cell cycle and down-regulated in nongrowing cells, as in the case of differentiated promyelocytic leukemia cells.