The packaging of the eukaryotic genome into chromatin is likely to regulate all processes that occur on the DNA template. The assembly and disassembly of chromatin structures from histone proteins and DNA are mediated by histone chaperones, including the histone H3/H4 chaperone anti-silencing function 1 (ASF1). To address the function of ASF1 in metazoan cells, we used RNA interference-mediated knockdown of Drosophila melanogaster ASF1 (dASF1). Cells lacking dASF1 accumulate in S phase of the cell cycle as determined by flow cytometry analysis of DNA content and quantitation of the proportion of cells with replication foci. In agreement, bromodeoxyuridine (BrdU) pulse-chase analysis demonstrates that the absence of ASF1 leads to delayed progression through S-phase. Furthermore, the absence of ASF1 leads to a reduced ability to incorporate the nucleoside analog BrdU, indicating that ASF1 is required for efficient DNA replication. We have also found that dASF1 colocalizes with DNA replication foci throughout S phase by immunofluorescence analysis and that these dASF1 foci are disrupted upon inhibition of DNA replication by treatment of cells with hydroxyurea. As such, these results demonstrate that dASF1 is present at active, but not stalled, replication forks. We propose that dASF1 has a direct role in modifying chromatin structure during DNA replication and that this function of dASF1 is important for the processivity of the replication machinery.