To understand the significance of p53 in relation to cell prolferation or cell cycle, we have studied the expression of p53 and PCNA or p53 in relation to DNA content using flow cytometry. Cell lines used for analysis were PCI-50, a human squamous cell carcinoma of the head and neck, SW-480, a human adenocarcinoma of the colon, and K562, a leukemia cell line. The cells were doubly-stained for p53(mouse anti-human p53-FITC(IgG2b, kappa)(DAKO)), and PCNA(mouse anti-human PCNA-RPE(IgG2a, kappa)(DAKO)), or p53 and DNA, and analyzed with FACStar plus(Beckton Dickinson). The fluorescence intensity of PCI-50 and SW-480 treated with the mouse anti-human p53-FITC was higher than in cells exposed to isotypic mouse IgG2b antibody, while K562 was not stained. In PCI-50 cell line, 10-40% of cells were positive with p53, while 50-80% positive in SW-480 cell line which was known to have a mutant-type p53. On the double staining of cell lines for p53 and PCNA, PCNA positive cells were divided to two subtypes, p53(+)/PCNA(+) and p53(-)/PCNA(+) and all-p53 positive cells were positive for PCNA. The p53(+)/PCNA(+) cells were distributed throughout the cell cycle in SW-480 cell line while p53 content in PCI-50 cell line begun to increase in S phase and accumulate in S and G2 phases during cell cycle progression. The results of this study suggest that expression of p53 protein in PCI-50 may be dependent on cell cycle and that mutant type p53 in SW-480 may be correlated with progression to cell cycle or may be constitutively expressed regardless of the cell cycle. The expression and role of p53 in cell cycle in vitro might be different among different cell lines. In addition to known S phase expression, PCNA might be also expressed in other phases of cell cycle.