Heterochromatin protein 1 (HP1) is a conserved nonhistone chromosomal protein with functions in euchromatin and heterochromatin. Here we investigated the diffusional behaviors of HP1 isoforms in mammalian cells. Using fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) we found that in interphase cells most HP1 molecules (50-80%) are highly mobile (recovery halftime: t(1/2) approximately 0.9 s; diffusion coefficient: D approximately 0.6-0.7 microm(2) s(-1)). Twenty to 40% of HP1 molecules appear to be incorporated into stable, slow-moving oligomeric complexes (t(1/2) approximately 10 s), and constitutive heterochromatin of all mammalian cell types analyzed contain 5-7% of very slow HP1 molecules. The amount of very slow HP1 molecules correlated with the chromatin condensation state, mounting to more than 44% in condensed chromatin of transcriptionally silent cells. During mitosis 8-14% of GFP-HP1alpha, but not the other isoforms, are very slow within pericentromeric heterochromatin, indicating an isoform-specific function of HP1alpha in heterochromatin of mitotic chromosomes. These data suggest that mobile as well as very slow populations of HP1 may function in concert to maintain a stable conformation of constitutive heterochromatin throughout the cell cycle.