Sister-chromatid cohesion, the machinery used in eukaryote organisms to prevent aneuploidy, tethers sister chromatids together after their replication in S phase until mitosis. Previous studies in fission yeast, Drosophila and mammals have demonstrated the requirement for the heterochromatin formation pathway for proper centromeric cohesion. However, the exact role of heterochromatin protein 1 (HP1) in sister-chromatid cohesion in mammals is still unknown. In this study, we disrupted endogenous HP1 expression in HeLa cells using a dominant-negative mutant of HP1beta and wild-type or mutant forms of HP1alpha. We then examined their effects on chromosome alignment, segregation and cohesion. Enforced expression of these constructs leads to frequent chromosome misalignment and missegregation. Mitotic chromosomes from these cells also exhibit a loosened primary constriction and separated sister chromatids. We further demonstrate that alignment of the cohesin proteins around kinetochores was also aberrant and that cohesin complexes bound less tightly in these cells. Unexpectedly, we observed a "wavy" chromosome morphology resembling that seen upon depletion of condensin proteins in cells with over-expression of HP1alpha, but not in cells expressing the HP1beta mutant. These results indicate that proper HP1 status is required for sister-chromatid cohesion in mammalian cells, and suggest that HP1alpha might be required for chromosome condensation.