Human amniotic fluid cells, known to express HLA-A, -B, and -C antigens, were tested for the presence of lymphocyte-stimulating antigens (LD or HLA-D) using modifications of the mixed lymphocyte culture (MLC) and primed lymphocyte typing (PLT) tests. Peripheral blood lymphocytes were co-cultured with various concentrations of allogeneic amniotic fluid cells, either growing as a monolayer culture in microtiter plates or suspended in medium following treatment with trypsin. The kinetics of such mixed lymphocyte amniotic fluid cell culture (MLAC) reactions were followed during days 3 to 8. Under none of these conditions did amniotic fluid cells significantly stimulate allogeneic lymphocytes, even after lymphocytes were specifically primed in the PLT assay to the HLA-D antigens segregating in the family of the amniotic fluid cell donor. Furthermore, in three-cell experiments, amniotic fluid cells failed to inhibit an ongoing MLC reaction, indicating that the absence of proliferative response to amniotic fluid cells is not due to active suppression. Taken together, these data strongly suggest that amniotic fluid cells either do not express HLA-D antigens or do not express them in a form that is detectable in either primary or secondary MLC.