The relative amounts of HLA-A,B,C antigens, beta 2-microglobulin (beta 2m), and trophoblast antigens (Trop-1 and Trop-2) were determined on nine choriocarcinoma cell lines including seven lines of gestational origin and two lines of nongestational origin (from ovary and stomach) by quantitative immunofluorescence analysis using a fluorescence-activated cell sorter. Most of these lines expressed surface HLA to variable extents, but one had none detectable. However, all lines secreted readily measurable amounts of beta 2m. We analyzed total RNA extracted from these lines using northern blot molecular hybridization with HLA-A,B,C- and beta 2m-specific complementary DNA probes. We found no messenger RNA species which hybridized with the HLA probe in cells with no detectable HLA surface antigen and only small amounts of HLA-specific RNA in cells with low levels of HLA membrane antigen. Cells exhibiting surface HLA levels greater than about 30% of that on lymphocytes had much higher amounts of HLA-specific RNA than did choriocarcinoma cells with no or low HLA antigen expression. In contrast, RNA hybridizing with beta 2m-specific probes was present at the 20% level or higher (relative to lymphocytes) in all the cell lines tested. Thus, the expression of HLA-A,B,C is apparently limited in choriocarcinoma cells by the level of HLA heavy-chain RNA and not by the level of beta 2m RNA. We discuss these findings in relation to the normal trophoblastic or other origins of this tumor type and with respect to the regulation and function of HLA in trophoblasts.