Human diploid fibroblasts (HDF) have a limited in vitro lifespan of population doubling level (PDL) 50-70. The molecular mechanism underlying cellular senescence and immortalization is not thoroughly understood. It has been reported that the cytoskeleton has diverse functions and may have a role in growth regulation through association with other cellular components. To shed light on the relationship between functions of the cytoskeleton and senescence or immortalization, we investigated the alterations in gene expression after immortalization and measured the amounts of mRNAs for talin, vinculin, alpha-actinin, tropomyosin 1 (TM1), vimentin, lamin A and C, and alpha-tubulin by slot blot and Northern blot analyses. We found that the mRNAs for vinculin and vimentin were reduced and the mRNA for lamin A was increased in immortalized cells. We also studied the cytoskeletal protein levels and their intracellular distributions by Western blot analysis and immunostaining. Most of the proteins studied behaved in a way similar to the mRNAs through senescence and immortalization. Vinculin, tropomyosins and vimentin showed their altered distributions in immortalized cells.