We previously reported that hairy cell leukemia (HCL) patients have high percentages of CD56+/CD57+/CD3+ large granular lymphocytes consistent with cytotoxic T-lymphocytes (CTLs), and other investigators have reported skewing of the T-cell repertoire. In previous studies of up to seven HCL patients, many of the 22 established T-cell receptor (TCR) beta variable region (TRBV) families showed mono- or oligoclonal restriction. To determine whether percentages of CTLs are correlated with TRBV clonal excess, we studied 20 HCL patients with flow cytometry, PCR of TCR gamma and TRBV regions, and fractional gel electrophoresis of PCR-amplified TRBV CDR3 domains (CDR3 spectratyping). Increased percentages of CD3+/CD8+/CD57+ CTLs correlated with more mono/oligoclonal and fewer polyclonal TRBV families (r=0.53; P=0.016). Age correlated with number of mono/oligoclonal TRBV families (r=0.51; P=0.022). Time since last purine analog therapy correlated with number of polyclonal TRBV families (r=0.46; P=0.040), but treatment with the anti-CD22 recombinant immunotoxin BL22 was not related to clonal excess. We conclude that abnormalities in the T-cell repertoire in HCL patients may represent deficient immunity, and may be exacerbated by purine analogs. Increased CD3+/CD57+ T-cells may be a useful marker of abnormal TRBV repertoire in HCL patients, and might prove useful in deciding whether patients should receive biologic antibody-based treatment rather than repeated courses of purine analog for relapsed disease.