Seven distinct anti-human T cell receptor (TcR) V region monoclonal antibodies (mAb) were generated by immunizing mice with either human T cell lines or transfected murine cells expressing human TcR V beta genes. The specificity of these reagents was determined as follows: T cells recognized by each mAb were purified from the peripheral blood of healthy donors and TcR transcripts expressed in these cells were analyzed using oligonucleotide-driven amplification and cDNA sequencing. Four mAb were found to delineate the V beta 3, V beta 8, V beta 17 and V beta 19 subfamilies, respectively. The remaining reagents recognize subsets within the V beta 2, V beta 5 and V beta 13 subfamilies. Reactivity of the mAb with circulating T cells from 18 unrelated healthy individuals was determined. Limited variability was found from an individual to another. In four donors, mAb staining was compared to oligonucleotide-driven amplification for evaluation of V beta 3, V beta 8, V beta 17 and V beta 19 subfamily expression in the peripheral blood. Although the V gene subfamily-specific oligonucleotides used in this study belong to a carefully controlled series, our results show that this method does not give an accurate estimate of the percentage of peripheral T cells expressing a given TcR beta chain. The present data confirm the necessity to establish a complete set of well-characterized monoclonal reagents to study human T cell responses.