We show here that treatment of HeLa cells with calyculin A, an inhibitor of Protein Phosphatases 1 and 2A, induces premature chromosome condensation (PCC) at any point in interphase of the cell cycle. Chromosomes in G(1)-phase PCC closely resemble metaphase chromatids in the light microscope, and measurements using FLIM-FRET show that they have the same level of chromatin compaction as metaphase chromosomes. However, histone H1 is not phosphorylated in G(1)- or early S-phase PCC. These results suggest that H1 phosphorylation is not required for mitotic chromosome condensation and chromatin compaction. They also confirm that Cdk1/cyclin B, which directly phosphorylates histone H1, is not active in G(1) and thus is not essential for G(1)-PCC. We suggest that induction of G(1)-PCC involves protein kinases or other factors that are either held in an inactive state by protein phosphatases, or constitutively active but countered by phosphatases. The same factors may be involved in the onset of normal mitosis, becoming active when protein phosphatases are downregulated. Induction of PCC with calyculin A should provide a useful system for identifying and studying the biochemical pathways that are required for mitotic chromosome compaction, nuclear envelope breakdown, and other events of mitosis.