Heterochromatin protein 1 (HP1) plays an important role in heterochromatin formation. Three subtypes of HP1, namely HP1alpha, beta, and gamma, 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 HP1beta localized most preferentially to centromeric heterochromatin, whereas HP1alpha and gamma were more preferentially localized to PML nuclear bodies. During metaphase, only HP1alpha, 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 HP1alpha was necessary for localization to the metaphase centromere and the N-terminal fragment (amino acid residues 1-76) of HP1beta was necessary for localization to the interphase centromere. Interestingly, simultaneous observations of residues 101-180 of HP1alpha and residues 1-76 of HP1beta in living HeLa cells revealed that during late prophase, the HP1beta fragment dissociated from centromeric regions and the HP1alpha fragment accumulated in centromeric regions. These results indicate that different specific regions of human HP1alpha and HP1beta 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.