The MCM protein family has been implicated in the regulatory machinery causing DNA to replicate only once in the S phase. Recently, we have identified human (h) CDC47, another member of the family, and demonstrated its unique S-phase nuclear association pattern implying participation in the regulation of DNA replication. In the present study, we applied an antisense strategy to assess the phenotype of human cells in which hCDC47 is repressed. As a result serum-induced accumulation of hCDC47 protein in human fibroblast cells was reduced by a specific antisense oligodeoxynucleotide and S-phase entry of such cells was significantly inhibited, indicating that hCDC47 protein is essential for DNA replication.