Cell cycle checkpoints are safeguards that ensure the initiation of downstream events only after completion of upstream processes. The tumor suppressors p53 and pRb prevent initiation of a second round of replication in response to spindle inhibitors, but it has yet to be proven that this is a mitotic checkpoint response. We show that asynchronous human fibroblasts arrest in G₁ with 4 N DNA content after nocodazole treatment, whereas isogenic p53- and pRb-deficient fibroblasts rereplicate. Importantly, nocodazole elicits a reversible arrest in G₀-G₁ synchronized normal human fibroblasts but not in isogenic p53-deficient derivatives. Furthermore, the G₁ cyclin-dependent kinase inhibitors p21 and p16 also play critical roles in limiting rereplication. Hence, p53 and pRb are required during G₁ to prevent entry into a replicative cycle and appear to provide a connection between the structural integrity of the microtubules and the cell cycle machinery in interphase cells.