Little is known about the gamma delta T cells in human neonatal umbilical cord blood. To compare neonatal cord blood and adult blood gamma delta T cells, we studied the V gamma and V delta gene segment usage in these populations by flow cytometry, and we derived cord blood gamma delta T cell clones to determine their functional capabilities. Unlike adult blood gamma delta T cells that predominantly express V gamma 2V delta 2 TCRs, neonatal cord blood gamma delta T cells expressed diverse V gamma and V delta gene segments paired in a variety of combinations rarely observed in adults. gamma delta T cell clones derived from neonatal cord blood similarly expressed a diverse array of TCRs. These cord blood-derived gamma delta T cell clones had weak cytolytic activity when assayed for K562 tumor cell killing, lectin-mediated cytolysis, and redirected cytolysis. They also expressed lower levels of the CD2, LFA-1, and CD45RO cell surface receptors as compared with strongly cytolytic adult gamma delta T cell clones. These properties of the cord blood-derived gamma delta T cell clones, weak cytotoxic activity and low adhesion molecule expression, were similar to the properties of the CD4+ subset of adult gamma delta T cells. Thus, neonatal gamma delta T cells are functionally different from the majority of adult gamma delta T cells and display a distinct TCR repertoire and accessory molecule profile.