CD3- leukocyte clones derived from human decidualized endometrial tissue of first trimester pregnancy have been compared with CD3- PBL clones. Most CD3- decidual granulated leukocyte (DGL) clones were CD16- CD56+, whereas most CD3- PBL clones were CD16+ CD56+. CD3- DGL and PBL clones, whether CD16+ or not, showed MHC-nonrestricted NK cell activity. However, CD3- CD16- DGL clones had low cytotoxic activity against the NK-resistant cell line BSM, whereas CD3- CD16+ DGL and CD3- PBL clones were strongly cytotoxic. Cytolytic activity has also been investigated in respect of target cell HLA-G expression, because this nonpolymorphic class I MHC molecule is expressed selectively by invasive fetal cytotrophoblast. Class I HLA Ag loss cell mutants were killed efficiently by CD3- DGL clones. Expression of transfected HLA-B8 increased their sensitivity to lysis by most CD3- DGL clones, whereas expression of transfected HLA-G commonly led to decreased target cell killing. In addition, the effects of uncloned CD3- DGL on the one-way MLR have been examined. These cells were very poor responders and, unless cultured to induce expression of class II MHC molecules, were also very poor stimulators. When fresh CD3- DGLs were added as third-party cells, either autologous or allogeneic to responder cells, [3H]TdR incorporation was decreased in the MLR. Thus, CD3- DGL clones express MHC-nonrestricted cytolytic activity, notably against HLA-negative cells, but expression of HLA-G offers protection to target cells. In addition, CD3- DGL may function to suppress allogeneic responses.