Heterochromatin protein 1 (HP1) plays an important role in heterochromatin formation. Three subtypes of HP1, namely HP1α, β, and γ, have been identified in humans. In this study, using yellow fluorescent protein(YFP) fusion constructs, we examined the intracellular localization of human HP1 subtypes during the cell cycle. During interphase, all three HP1 subtypes were localized to centromeric heterochromatin and to promyelocytic leukemia(PML) nuclear bodies. Different preferences, however, were observed among the subtypes: during interphase HP1β localized most preferentially to centromeric heterochromatin, whereas HP1α and γ were more preferentially localized to PML nuclear bodies. During metaphase, only HP1α, was localized to the centromere. We thus determined which molecular domains of HP1 were necessary for their intracellular localization. Our results showed that the C-terminal fragment (amino acid residues 101-180)of HP1α was necessary for localization to the metaphase centromere and the N-terminal fragment (amino acid residues 1-76) of HP1β was necessary for localization to the interphase centromere. Interestingly, simultaneous observations of residues 101-180 of HP1α and residues 1-76 of HP1βin living HeLa cells revealed that during late prophase, the HP1βfragment dissociated from centromeric regions and the HP1α fragment accumulated in centromeric regions. These results indicate that different specific regions of human HP1α and HP1β mediate localization to metaphase and interphase centromeric regions resulting in association of different subtypes of HP1 with the centromere at different times during the cell cycle.