The α chain of the human histocompatibility antigen HLA-G was identified as an array of five 37- to 39-kilodalton isoforms by the use of two-dimensional gel electrophoresis. Both cell-associated and secreted HLA-G antigens are prominent in first trimester villous cytotrophoblasts and are greatly reduced in third trimester cytotrophoblasts. Allelic variation was not detected, an indication that HLA-G is not obviously polymorphic in cytotrophoblasts. Among the following choriocarcinoma cell lines studied, HLA-G is expressed in JEG but not in Jar or BeWo. Expression of endogenous HLA-G genes has not been found in normal lymphoid cells. Thus, HLA-G is subject to both cell type-specific and developmental regulation and is expressed in early gestation human cytotrophoblasts.