Cell division in the absence of telomerase causes progressive telomere shortening which ultimately leads to telomere dysfunction and initiation of genome instability. In order to identify factors related to loss of telomere function, the effects of telomerase inhibition on the proteome of five tumor cell lines were followed by SELDI-TOF-MS. Five differentially expressed protein peaks (p<0.01) were found in a total of 60 clones of five cell lines representing four tissues (lung, breast, prostate, and colon) in which telomerase was inhibited by retroviral overexpression of a dominant negative (DN) mutant of human telomerase reverse transcriptase (hTERT). Among these, a 11.3 kDa peak diminished in DN-hTERT clones was identified as histone H4 by nanoflow-HPLC-MS/MS. Immunoblot analysis not only confirmed the decline of histone H4, but also of other core histone proteins including histone H3. Furthermore, upregulation of several cytokeratins was found to be associated with telomere attrition. In conclusion, loss of telomere function is associated with alterations in the proteome which may represent novel biomarkers for the detection of replicative senescence.