We have investigated the regulation of cell-cycle entry in C. elegans, taking advantage of its largely invariant and completely described pattern of somatic cell divisions. In a genetic screen, we identified mutations in
cyd-1 cyclin D and
cdk-4 Cdk4/6. Recent results indicated that during Drosophila development, cyclin D-dependent kinases regulate cell growth rather than cell division. However, our data indicate that C. elegans
cyd-1 primarily controls G1 progression. To investigate whether
cyd-1 and
cdk-4 solely act to overcome GI inhibition by retinoblastoma family members, we constructed double mutants that completely eliminate the function of the retinoblastoma family and cyclin D-Cdk4/6 kinases. Inactivation of
lin-35 Rb, the single Rb-related gene in C elegans, substantially reduced the DNA replication and cell-division defects in
cyd-1 and
cdk-4 mutant animals. These results demonstrate that
lin-35 Rb is an important negative regulator of G1/S progression and probably a downstream target for
cyd-1 and
cdk-4. However, as the suppression by
lin-35 Rb is not complete,
cyd-1 and
cdk-4 probably have additional targets. An additional level of control over G1 progression is provided by Cip/Kip kinase inhibitors. We demonstrate that
lin-35 Rb and
cki-1 Cip/Kip contribute nonoverlapping levels of G1/S inhibition in C. elegans. Surprisingly, loss of
cki-1, but not
lin-35, results in precocious entry into S phase. We suggest that a rate limiting role for
cki-1 Cip/Kip rather than
lin-35 Rb explains the lack or cell-cycle phenotype of
lin-35 mutant animals.