Substantial numbers of both alpha/beta and gamma/delta T cells are present in human fetal liver, which suggests a role of the fetal liver in T cell development. The diversity of fetal liver T cell receptor (TCR) gamma and delta chain rearrangements was examined among both CD4+CD8- and CD4-CD8- gamma/delta T cell clones. In addition, TCR delta chain transcripts from three fetal livers were sequenced after polymerase chain reaction amplification of TCR delta chains with V delta 1 or V delta 2 rearrangements. Five of six fetal liver gamma/delta T cell clones had a V delta 2-D delta 3-J delta 3 gene rearrangement with limited junctional diversity; three of these clones had an unusual CD4+CD8- phenotype. V delta 2-D delta 3-J delta 3 gene rearrangements were also common among both in-frame and out-of-frame transcripts from three fetal livers, indicating that they are the result of an ordered rearrangement process. TCR gamma chain sequences of the fetal liver gamma/delta T cell clones revealed V gamma 1-J gamma 2.3, V gamma 2-J gamma 1.2, and V gamma 3-J gamma 1.1 rearrangements with minimal incorporation of template-independent N region nucleotides. TCR gamma chain rearrangements found in these fetal liver T cell clones were different from those that have been observed among early thymic gamma/delta T cell populations, while similar TCR delta chain rearrangements are found among gamma/delta T cells from both sites. These data demonstrate that the fetal liver harbors gamma/delta T cell populations distinct from those found in the fetal thymus, suggesting that the fetal liver is a site of gamma/delta T cell development in humans. These unusual T cell populations may serve a specific function in the fetal immune system.