To become mature alphabeta T cells, developing thymocytes must first assemble a T cell receptor (TCR) beta chain gene encoding a TCRbeta chain that forms a pre-TCR. These cells then need to generate a TCRalpha chain gene encoding a TCRalpha chain, which, when paired with the TCRbeta chain, forms a selectable alphabeta TCR. Newly generated VJalpha rearrangements that do not encode TCRalpha chains capable of forming selectable alphabeta TCRs can be excised from the chromosome and replaced with new VJalpha rearrangements. Such replacement occurs through the process of TCRalpha chain gene revision whereby a Valpha gene segment upstream of the VJalpha rearrangement is appended to a downstream Jalpha gene segment. A multistep, gene-targeting approach was used to generate a modified TCRalpha locus (TCRalpha(sJ)) with a limited capacity to undergo revision of TCRalpha chain genes. Thymocytes from mice homozygous for the TCRalpha(sJ) allele are defective in their ability to generate an alphabeta TCR. Furthermore, those thymocytes that do generate an alphabeta TCR have a diminished capacity to be positively selected, and TCRalpha(sJ/sJ) mice have significantly reduced numbers of mature alphabeta T cells. Together, these findings demonstrate that normal T cell development relies on the ability of developing thymocytes to revise their TCRalpha chain genes.