CD8lo4lo cells are the immediate precursors of immature CD8hi4loTcRlo, CD8lo4hiTcRlo and CD8hi4hiTcRlo double-positive (DP) thymocytes in the adult murine thymus. These cells are the first subset in the adult thymus to express accessory CD8 and CD4 molecules, to rearrange the T cell receptor (TcR) alpha chain genes and to express the TcR alpha beta heterodimer at low levels at the surface. Here, we investigate the fetal ontogeny of CD8lo4lo cells. We detect these cells on day 15 of fetal development. They dominate the thymus on day 15.5, to become progressively less prominent thereafter. An important characteristic of fetal CD8lo4lo cells is the early expression of TcR alpha mRNA (on fetal day 15, 36-48 h earlier than reported previously). Our results also suggest, but do not prove, that the receptor may be expressed on the surface as early as day 15.5. Fetal CD8lo4lo cells, like their adult counterparts, become DP in vitro. However, early fetal CD8lo4lo thymocytes express both CD44 and CD25--unlike the adult subset--and that links them to their putative precursors, fetal CD44+CD25+ double-negative cells. This finding underscores the difference between adult and fetal thymocytes in turnover of membrane molecules and/or the kinetics of progression through phenotypic stages.