Summary— Specific antibodies were prepared against Drosophila DNA polymerase e and DREF, a regulatory factor for DNA replication‐related genes. Using these antibodies together with those for DNA polymerase α and proliferating cell nuclear antigen (PCNA), we examined expression patterns and sub‐cellular distributions of these proteins during Drosophila development. DNA polymerase α, ε and PCNA proteins were maternally stored in unfertilized eggs and maintained at high levels during embryogenesis. With distinct nuclear localization, proteins were observed in embryos at interphase stages throughout the 13 nuclear division cycles, suggesting that they all participate in rapid nuclear DNA replication during these cycles. In contrast, maternal storage of a DREF protein was relatively low and its level increased throughout embryogenesis. Strong nuclear staining with the anti‐DREF antibody was not observed until the nuclear division cycle 8. Immunostaining of various larval tissues from transgenic flies carrying the PCNA gene promoter‐lacZ fusion gene revealed co‐expression of DREF, PCNA and lacZ, suggesting that DREF regulates the expression of PCNA gene in these tissues. In addition, we detected a relatively high level of DREF in adult males as well as females. Since DNA polymerase α, ε and PCNA are hardly detectable in adult males, DREF very likely regulates genes other than those closely linked to DNA replication in adult males.