Anti-T cell receptor (aTCR) antibody (Ab) stimulation of T cells results in TCR down-modulation and T cell activation. Differences in the effect of anti-alpha-chain and beta-chain Ab have been reported on thymocytes. Anti-beta-chain Ab but not anti-alpha-chain reagents cause long-term TCR down-modulation. However, both types of Ab result in TCR cross-linking and activate early steps in signal transduction. In this study, we show that TCR internalization and calcium flux, hallmarks of T cell activation, are similar with aValpha and aVbeta treatment. Therefore, we have compared the gene expression profiles of preselection thymocytes stimulated with these reagents. We find that aValpha treatment does not cause any significant change in gene expression compared with control culture conditions. In contrast, aVbeta stimulation results in numerous changes in gene expression. The alterations of expression of genes known to be expressed in thymocytes are similar to changes caused by positive thymic selection, suggesting that the expression of some of the genes without known roles in thymocyte development and of novel genes whose expression is found to be altered may also be involved in this process.