AN intact nuclear membrane restricts DNA replication to only one round in each cell cycle, apparently by excluding an essential replication-licensing factor throughout interphase(1-5). A family of related yeast replication proteins, MCM2, 3 and 5 (also called, after cell-division cycle, CDC46), resemble licensing factor, entering the nucleus only during mitosis(6-8). We have cloned a Xenopus homologue of MCM3 (XMCM3) and raised antibodies against expressed protein, Immunodepletion of Xenopus egg extracts removes a complex of MCM2, 3 and 5 homologues and inhibits replication of Xenopus sperm nuclei or permeable G2 HeLa nuclei. However, G1 HeLa nuclei still replicate efficiently. Mock-depleted extracts replicate all three templates. XMCM3 accumulates in nuclei before replication but anti-XMCM3 staining decreases during replication. These results can explain why replicated nuclei are unable to reinitiate replication in a single cell cycle.