The expression of minor lymphocyte stimulatory locus (Mls) determinants in combination with murine major histocompatibility complex (MHC) class II molecules, leads to the destruction of lymphocytes bearing specific V region-encoded T cell receptor (TcR) products. A much studied example is the elimination of V beta 6+ cells in IE+/Mls-1a mice, in which deletion can be detected 7-10 days after birth but is not fully operational earlier in embryonic life. Here we investigate this transitional period in development and show that selective deletion of V beta 6 occurs in vitro, approximately 1 week after organ cultures are established from 14 day embryos. These unmanipulated organ cultures receive no additional cell immigrants after day 14, suggesting that the cellular elements mediating negative selection (or their direct precursors), are already resident in the fetal thymus by day 14 of gestation. Hence, the developmental timing of the outset of rigorous negative selection of V beta 6 is not dictated by the postnatal entry of deleting elements into the thymus, but perhaps by the maturation of the pre-existing environment. Using a parallel organ-culture approach we have looked at the development of V delta 4 and V gamma 3, TcR gamma delta+ cells in a variety of mouse strains. These receptors have recently been reported to be subject of MHC and non-MHC linked selection, respectively. We find that after an initial period of expansion, the number of V gamma 3-expressing cells dramatically declines. However, this selective loss of V gamma 3 cells is not contingent on the C57BL/6 mouse strain (in contrast to a previous report). These findings are discussed in the context of current models of ontogeny and repertoire selection.