Geminin is an unstable regulatory protein that affects both cell division and cell differentiation. Geminin inhibits a second round of DNA synthesis during S and G2 phase by binding the essential replication protein Cdt1. Geminin is also required for entry into mitosis, either by preventing replication abnormalities or by down-regulating the checkpoint kinase Chk1. Geminin overexpression during embryonic development induces ectopic neural tissue, inhibits eye formation, and perturbs the segmental patterning of the embryo. In order to define the structural and functional domains of the geminin protein, we generated over 40 missense and deletion mutations and tested their phenotypes in biological and biochemical assays. We find that geminin self-associates through the coiled-coil domain to form dimers and that dimerization is required for activity. Geminin contains a typical bipartite nuclear localization signal that is also required for its destruction during mitosis. Nondegradable mutants of geminin interfere with DNA replication in succeeding cell cycles. Geminin's Cdt1-binding domain lies immediately adjacent to the dimerization domain and overlaps it. We constructed two nonbinding mutants in this domain and found that they neither inhibited replication nor permitted entry into mitosis, indicating that this domain is necessary for both activities. We identified several missense mutations in geminin's Cdt1 binding domain that were deficient in their ability to inhibit replication yet were still able to allow mitotic entry, suggesting that these are separate functions of geminin.