We have identified a large population of CD3(-)7(+) cells in human fetal gut. Three- and four-color flow cytometry revealed a distinct surface Ag profile on this population; the majority were negative for CD4 and CD8, whereas most of the remainder expressed the CD8alphaalpha homodimer. In contrast about half of CD3(+) cells expressed CD4 and half expressed CD8alpha. A large proportion of CD3(-)7(+) cells expressed CD56, CD94, and CD161, and whereas CD3(+) T cells also expressed CD161, they only rarely expressed CD56 or CD94. Further studies were conducted to determine whether the CD3(-)7(+) cells have the potential to differentiate into CD3(+) cells. About half of CD3(-)7(+) cells contain intracellular CD3epsilon. Rearranged TCR gamma-chains were detected in highly purified CD3(-)7(+) cells as an early molecular sign of T cell commitment, and the pattern of rearrangement with V regions spliced to the most 5' Jgamma segment is reminiscent of early thymocyte differentiation. In reaggregate thymic organ cultures, CD3(-)7(+) cells also gave rise to CD3(+) T cells. Thus, we demonstrate that the CD3(-)7(+) cells present in the human fetal gut display a distinct phenotype and are able to develop into CD3(+) T cells.