Multiparameter-flow-cytometry was used to compare the levels of two proliferation-associated antigens, proliferating cell nuclear antigen (PCNA) and the Ki-67-related antigen, in squamous carcinoma cells grown as monolayers, multicellular spheroids (MCTS), and xenograft tumors. While the level of Ki-67-positive cells decreased with time of culture, the percentage of PCNA-positive cells stayed high in all experimental states investigated (up to 5 weeks of growth). The reduction of the mean fluorescence of PCNA/cell indicated a different way of regulation of this antigen in squamous carcinoma cells in comparison to other cell types.