During Drosophila eye development, cell proliferation is coordinated with differentiation. Immediately posterior to the morphogenetic furrow, cells enter a synchronous round of S phase called second mitotic wave. We have examined the role of RBF, the Drosophila RB family homolog, in cell cycle progression in the second mitotic wave. RBF-280, a mutant form of RBF that has four putative cdk phosphorylation sites mutated, can no longer be regulated by Cyclin D or Cyclin E. Expression of RBF-280 in the developing eye revealed that RBF-280 does not inhibit G1/S transition in the second mitotic wave, rather it delays the completion of S phase and leads to abnormal eye development. These observations suggest that RB/E2F control the rate of S-phase progression instead of G1/S transition in the second mitotic wave. Characterization of the role of RBF in Cyclin D/Cdk4-mediated cellular growth showed that RBF-280 blocks Cyclin D/Cdk4 induced cellular growth in the proliferating wing disc cells but not in the non-dividing eye disc cells. By contrast, RBF-280 does not block activated Ras-induced cellular growth. These results suggest that the ability of Cyclin D/Cdk4 to drive growth in the proliferating wing cells is distinct from that in the none-dividing eye cells or the ability of activated Ras to induce growth, and that RBF may have a role in regulating growth in the proliferating wing discs.