Although it is well known that the expression of major histocompatibility complex (MHC) antigens on the surface of lymphoblastoid cell lines are cell cycle dependent, the way in which the MHC antigen expression on activated T cells varies with cell cycle phase has not previously been described. Using 11 lymphoblastoid cell lines from malignant and nonmalignant tissues (B cells, T cells, and myeloid cells) and five activated T cell lines (two cell lines activated by phytohemagglutinin and three alloreactive T cell clones), MHC antigen expression was quantitatively studied by dual-beam flow cytometry. Correlated measurements of surface antigen quantity (immunofluorescence), DNA content (Hoechst 33342), and cell size (light scatter), uninfluenced by induction synchrony and cell fixation, were performed. The data indicate that cell surface antigen quantity and cell surface area demonstrate specific values at each phase of the cell cycle when the cells are in logarithmic growth. Examining cells in logarithmic growth, it was confirmed, for all lymphoblastoid cell lines, that the quantity of MHC antigens on G2 (S + G2 + M) cells was greater than that on G1 cells. In addition, it was found, by analyzing antigen quantity and surface area, that class I antigen density in the G2 phase is 17% less than that in the G1 phase in leukemic T cell lines, and that both class I and class II antigen densities in the G2 phase were 21% less than that in the G1 phase in lymphoblastoid B cell lines. In activated T cells, class I antigen density in the G2 phase was 11% less than that in the G1 phase, while class II antigen density in the G2 phase was 12% greater than that in the G1 phase. We describe four important observations in this report. In both G1 and G2 phases, activated T cells express: quantitatively fewer class I antigens than lymphoblastoid B cell lines; similar quantity of class I antigens as that of leukemic T cell lines; and similar quantity of class II antigens as that of lymphoblastoid B cell lines. Also, class II antigens are expressed in greater density in the G2 phase than in the G1 phase in activated T cells. In contrast, lymphoblastoid B cell lines express greater density of class II antigens in the G1 phase than in the G2 phase of the cell cycle. These findings differ from previous reports, suggesting that G1 phase cells may have a more significant role than G2 phase cells as target cells for MHC restricted cytotoxic cells.