T cell receptor Vβ gene usage in renal allograft-derived T cell lines was investigated using a semiquantitative, polymerase chain reaction (PCR)-based technique. A panel of Vβ-specific primers was carefully designed to have uniform hybridization characteristics, enhancing comparability, and high specificity based on 3′ mismatches between primers and nontarget V regions. PCR data were in concordance with previous phenotypic, functional, and Southern blot analyses, but the PCR technique proved to be more sensitive and comprehensive. For example, the EH3 cell line was confirmed to be polyclonal, with a number of different Vβ genes mediating anti-DR8-linked reactivity. Sequence analysis showed the most prevalent signals obtained by PCR, those for Vβ7 and Vβ20, probably corresponded to the only two rearrangements detected by Southern. The J2 cell line was shown to be polyclonal in early culture, when it was mostly CD8+ and B35-reactive. Late cultures and a subclone were CD4+, possessed DR3-linked reactivity, and evidenced only one PCR signal: Vβ6. Similarly, the MH3 cell line was shown to be polyclonal early in culture, when it was reactive toward All and A29. In late cultures, when Southern analysis indicated clonality, only anti-All reactivity was maintained, and the predominant PCR signal was for Vβ17. Other alloreactivities were also attributed to specific Vβ. The Kng cell line, autoreactive to A28, yielded two strong PCR signals; Vβ2 and Vβ9. The Mijo line, DR1-reactive, gave a predominant signal for Vβ12. Thus in spite of variable in vitro selection occurring in some lines and great initial heterogeneity documented by PCR, this technique was still capable of identifying Vβ genes that persist in vitro, become predominant, and are associated with specific, allograft-directed alloreactivities.