We investigated whether fetal mouse T cell receptor (TCR) gamma delta cells have been subjected to so-called TCR beta selection at the CD25 stage of thymus development. To this end, we carried out a comparative three-color flow microfluorimetric analysis to TCR gamma delta cells developing in the fetal, neonatal and adult thymus using monoclonal antibodies to CD2, CD8, CD24, CD25 and CD44. Day-15 fetal TCR gamma delta cells were CD2+ suggesting an origin at a post-CD25 stage. Molecular analysis of TCR beta rearrangements were also carried out. Thus, by semi-quantitative polymerase chain reaction (PCR) amplification of V beta 6 and V beta 8 to J beta 2 rearrangements day-15 fetal TCR gamma delta showed extensive TCR beta rearrangements, a finding confirmed by PCR amplification from single micromanipulated cells. Finally, sequencing analysis of 104 PCR-amplified TCR VDJ beta 2 fragments showed that the majority (58%) were rearranged out of frame . Taken together, these phenotypic and molecular analyses suggest that fetal TCR gamma delta cells have not been subject to TCR beta selection.