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We have developed an adaptor ligation PCR-based microplate hybridization assay (MHA) for analysis of T cell receptor alpha chain variable region (TCRAV) and T cell receptor beta chain variable region (TCRBV) repertoires. Forty three TCRAV and thirty eight TCRBV-specific probes were immobilized onto microplate wells in water-soluble carbodiimide. After hybridization of 5'-biotinylated PCR products, quantitative ELISA was carried out and followed by automated colorimetric reading. The conditions for immobilization and hybridization were optimized using representative TCRBV-specific probes. The sensitivity of MHA allows us to detect as low as 40 pg of biotinylated PCR products. The frequencies of individual V segments obtained by MHA were consistent with those obtained by FACS analysis and reverse dot blot assays. Analysis of the entire TCRAV and TCRBV repertoires could be done using a single 96-well plate, and completed in less than 6 h. Simplicity and reproducibility of this method make it suitable for routine laboratory use. The expression of TCRAV and TCRBV segments was next studied in peripheral blood mononuclear cells (PBMC) of 14 healthy donors using the newly developed MHA method. TCRAV8S1, TCRAV23S1, TCRBV2S1, TCRBV3S1, TCRBV4S1, and TCRBV6S5 were highly expressed in PBMC. Further, the TCRAV repertoires among individuals were less variable compared to the TCRBV repertoires. Interestingly, considerable variations in the expression levels of BV3S1, BV4S1, and BV17S1 were observed among individuals. One polymorphic site was found at the coding region of BV4S1, and there were two alleles. These results suggest that variable expression among individuals may be associated with unknown allelic polymorphism in coding and/or regulatory regions of these TCRBV segments, or with disparity in HLA genes.