We have recently demonstrated a remarkable selection of in vitro cultivated, TNP-specific polyclonal T cell lines for the expression of a TCR beta chain encoded by the V beta 8.2 gene. The goal of the present study was to analyse V alpha usage in V beta 8.2 T cells responsive to TNP, using TNP-specific T cell lines derived from three common strains of mice, as well as from V beta 8.2 transgenic mice. Results indicate that in vitro TNP stimulation of T cells from TNP-immune mice results in significant skewing of V alpha usage among responding V beta 8.2+ T cells, with overexpression observed for V alpha 3.2 and V alpha 8. These results indicate that V alpha expression influences recognition of TNP by T cells, and suggest that the hapten TNP might be recognized like typical peptide antigens by combinatorial TCR alpha and beta contact sites.