Gadd45a is a p53-regulated gene whose protein product, like p53, is involved in maintenance of genome stability. Specifically, deletion of Gadd45a leads to extensive aneuploidy as a consequence of centrosome amplification and subsequent abnormal segregation of chromosomes during mitosis. S-phase checkpoints were investigated in Gadd45a(-/-) cells to determine possible defects contributing to the uncoupling of centrosome duplication and DNA replication. In the presence of hydroxyurea, Gadd45a(-/-) mouse embryo fibroblasts show increased centrosome amplification coupled with loss of a sustained S-phase checkpoint. Gadd45a deletion allows another form of genomic instability, gene amplification, when p21 (Cdkn1a gene product) is deleted also. Gene amplification in Gadd45a(-/-)p21(-/-) cells correlated with loss of both G(1) and S-phase checkpoints. Multiple conditions of nutrient deprivation failed to prevent DNA synthesis in Gadd45a(-/-) cells. Gadd45a is therefore required for proper S-phase control and checkpoints under multiple conditions of nutrient deprivation. It is proposed that loss of S-phase control may account for both the uncoupling of DNA replication and centrosome duplication, and conferring gene amplification proficiency in cells lacking Gadd45a(-/-). This is of particular importance for solid tumors, which may lack sufficient nutrients yet are unable to elicit checkpoints preventing genomic instability under these conditions.