Aplastic anemia (AA) is characterized by multilineage bone marrow failure of unknown etiology. In order to assess the role of immune-mediated mechanisms in hematopoietic suppression, we examined the diversity of T lymphocyte repertoire in terms of variable (V) gene segment usage of the T cell receptor (TCR) beta chain in bone marrow and peripheral blood of six patients with severe untreated AA. Expression of transcripts encoding Vbeta1-Vbeta24 subfamilies was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The results revealed that T lymphocytes in AA utilize highly diverse segments of the beta chain loci. Over the heterogenous Vbeta expression background, transcripts encoding Vbeta3, Vbeta20, Vbeta21, and Vbeta22 subfamilies were enhanced by at least threefold in 5 of 6 patients as compared to normal samples, but a different transcript species was over expressed in each patient. To evaluate clonality of T cells, size diversity within the complementarity determining region 3 (CDR3) and usage of TCRbeta joining (J) gene segments were analyzed in PCR products specific for each of the 24 Vbeta subfamilies. We found that the majority of transcripts display normal CDR3 size patterns, as is characteristic of polyclonal populations. Nevertheless, one or two predominating junctional rearrangements were observed in each patient. They were identified in Vbeta5, Vbeta7, Vbeta8, Vbeta13, Vbeta15, Vbeta16, and Vbeta23 transcripts, which differed from patient to patient and did not correspond to transcripts with an abnormally high expression level. Our results demonstrate that T cell repertoire in AA is random with respect to the TCR beta chain. Unique rearrangements detected in the CDR3 region are suggestive of a limited process of an antigen-driven (oligo)clonal T cell expansion which may take place over the overwhelmingly polyclonal repertoire of T lymphocytes at the onset of severe AA.