We recently showed that circulating human CD8⁺ effector cells have a CD45RA⁺CD27^– membrane phenotype. In itself this phenotype appeared to pose a paradox: CD45RA, a marker expressed by unprimed cells, combined with absence of CD27, characteristic for chronically stimulated T cells. To investigate whether differentiation towards the CD45RA⁺CD27^– phenotype is dependent on antigenic stimulation and involves cellular division, TCR V_β usage and telomeric restriction fragment (TRF) length were analyzed within distinct peripheral blood CD8⁺ subsets. FACS analysis showed that the TCR V_β repertoire of CD8⁺CD45RA⁺CD27^– cells differed significantly from that of unprimed CD8⁺CD45RA⁺CD27⁺ cells. Moreover, in two out of six individuals large expansions of particular V_β families were observed in the CD8⁺CD45RA⁺CD27^– subset. CDR3 spectrotyping and single-strand confirmation analysis revealed that within the CD8⁺CD45RA⁺CD27^– population most of the 22 tested V_β families were dominated by oligoclonal expansions. The mean TRF length was found to be 2.3 ± 1.0 kb shorter in the CD8⁺CD45RA⁺CD27^– subset compared with the unprimed CD8⁺CD45RA⁺CD27⁺ population, but did not differ substantially from that of memory type, CD8⁺CD45RA^–CD27⁺ T cells. These findings indicate that the CD8⁺CD45RA⁺CD27^– cytotoxic effector population consists of antigen-induced, clonally expanded cells and confirm that the expression of CD45RA is not a strict marker of antigen non-experienced T cells.