Natural killer (NK) cell phenotype and activity was studied by analyzing uncultured and short-time-cultured murine NK cells from fetal day 17 spleen and thymus. In contrast to NK cells from adult mice, freshly sorted fetal NK cells did not contain NK receptor transcripts for Ly-49A, B, C/I, D, F, G2, or H. The only NK receptor transcripts that could be detected were Ly-49E and CD94. It is important that Ly-49E was present at a 10- to 30-fold higher level compared with uncultured NK cells from adult mice. After short-time interleukin-2 culture, the level of Ly-49E mRNA was comparable between fetal and adult NK cells. Functionally, fetal NK cells only killed MHC class I-negative tumor cells when activating NK receptors were cross-linked with antibody. We show that fetal NK cells are mature but are different from NK cells in adult mice regarding their NK cell receptor repertoire and function.