Differential screening-selected gene aberrative in neuroblastoma (DAN) gene (previously named N03 gene), whose expression is significantly reduced in transformed cells, has recently been demonstrated to have a tumor-suppressive activity in vitro. In order to investigate biological roles of DAN gene product in normal rat fibroblasts (3Y1), marker-selected transfectants that expressed the high amount of DAN gene product were generated from 3Y1 cell lines. These clones did not exhibit morphological changes compared with parental 3Y1 cells; however, they showed a decrease in growth rate and a remarkable reduction in saturation density. Cell cycle analysis revealed that the overexpression of DAN gene product causes the retardation of the entry into the S phase. These results suggest that DAN gene product may have an important role in regulation of the entry of cells into the S phase.