Interleukin-2 (IL-2) generates T lineage cell lines (IL-3B series) directly from the isolated interleukin 3 (IL-3)-induced pluripotential progenitor colonies obtained form adult spleen in vitro. The phenomenon can be reproduced by the combined IL-3/IL-2 bulk culture system. In order to compare the differentiation potential of such progenitors in various ontogenical stages, we have established a number of cell lines from fetal livers (LFD series), fetal thymus (FTD15), newborn spleens (SED series), and adult spleens (SPB series) using the IL-3/IL-2 culture system. The phenotypes of cell lines varied depending upon the developmental stages; LFD lines, Thy1+CD3−CD4−CD8−B22O+ FTD15, Thy1+CD3+CD4−CD8+ B220−, and SPB and IL-3B lines, Thy1+ CD3+CD4−CD8+B220+. Analysis of T cell receptor (TCR) genes revealed that all of the TCR genes (α, β, γ, and δ) were in germ line configurations in LFD lines. In LFD17 and 19, from 17 and 19 gestation days (GD) respectively, however, germ line transcripts or TCR β gene were detected, suggesting that they were in the earliest phase of T cell commitment. In LFD14 line from GD14 fetal liver, no such transcript could be detected. In contrast, FTD15 showed complete rearrangement of every TCR gene with full-length TCR α andβ gene mRNA. On the other hand, all of the SED, SPB, and IL-3B series of lines exhibited the rearrangement of every TCR gene with productive TCR α andβ gene mRNA. The results indicated that progenitors in postnatal spleen, in contrast to those in fetal liver, had the potential to rearrange and express the TCR genes in extrathymic conditions in vitro. Differences were also observed in IL-2R complex between LFD lines and other lines from postnatal spleens. Taken together, these results suggested that IL-3-responsive progenitor cells in various developmental stages had distinct IL-2-induced differentiation potential into T cell-lineage in vitro. Possible physiological implications of the results in the in vivo formation of T cell compartment are discussed.