Understanding factors regulating expression of tumor antigens is vital to the design of rational immunotherapeutic strategies. A murine monoclonal antibody (MM2-3C6) that recognizes a B16 murine melanoma-associated membrane antigen was used to study antigen expression in relation to cell cycle, clonal heterogeneity, and growth density. Dual-parameter flow cytometric measurements of DNA content and membrane antigen demonstrated that antigen-positive cells were found throughout the cell cycle. However, some antigen-negative cells were observed and were restricted to the G0/G1 phase demonstrating the antigenic heterogeneity of this tumor line. Three sublines of B16, F1, F10, and F1r with variable metastatic potential, and 50 B16 F1 clones all expressed the antigen with a mean antigen density above background of 8.84 +/- 2.52 [( mean cell fluorescence/cell size] X 10(-3] ranging from 3.68 to 16.57. Further studies of three sublines and five clones showed antigen density fluctuated over an 8-day growth period in culture with daily changes of medium. During log growth from Day 1 (1.2 +/- 0.08 X 10(4) cells/cm2) to Day 4 (20.4 +/- 3.01 X 10(4) cells/cm2), antigen density increased from 4.09 +/- 0.29 to 6.86 +/- 0.29. By Day 8 (26.5 +/- 2.86 X 10(4) cells/cm2) when the cells had been confluent for 3 days, antigen density decreased to 2.54 +/- 0.26 which was significantly lower than other days measured (P less than 0.05). It is concluded that tumor cell proliferation and cell density can modulate antigen expression in this system and, therefore, this may be a useful model to study tumor cell escape from immunotherapy.