The significant increase in chromosomal instability with aging is well known, but the underlying mechanism is not fully understood. Our earlier studies showed a high frequency of abnormal mitosis, such as mitotic slippage or incomplete mitosis in near-senescent human fibroblasts. This study examined the centrosome aberrations in mitotic and interphase cells from different passages of several strains of human fibroblasts. Analysis by laser scanning cytometry showed increased frequencies of abnormal mitotic cells with supernumerary (>2/cell) centrosomes and misaligned chromosomes in later passages in all strains examined. Numerical centrosome aberrations were prominent in a polyploid subpopulation. In metaphase cells, numerical centrosome aberrations were correlated significantly with chromosome misalignment. Fluorescent in situ hybridization analysis using a centromere-specific probe revealed a correlation between chromosome aneusomy and centrosome over-duplication. These results suggest that abnormal duplication of centrosomes in association with cellular aging may be responsible for the increase in chromosomal instability with aging.