OBJECTIVE To investigate the cell cycle expression of p53 protein, c-myc gene product and tyrosine phosphorylation level in human breast cancer cells. STUDY DESIGN Using a multifluorescence imaging procedure, the concentration per cell in different phases of the cell cycle can be evaluated by analyzing the bivariate contour plot of DNA content versus antigen concentration. RESULTS Low fluorescence intensity was observed in the G0/G1 phase for the three markers. The analysis of individual cells demonstrated that approximately 10% of cells were negative. During the G1/S transition, the fluorescence intensity of the three antigens increased rapidly. However, after the mild S-phase, the increase of c-myc was more marked than the tyrosine phosphorylation level, whereas p53 protein remained stable, with a slight tendency to decrease. CONCLUSION This study confirmed that the p53 protein and c-myc gene product could perform a regulatory function in G1/S transition and, consequently, may play an important role in malignant transformation. Like-wise, the variations of tyrosine kinase activity were linked to cellular progression throughout the cell cycle and could be a useful marker of alteration in the growth-factor signaling pathway. Thus, the multifluorescence imaging procedure may provide useful information on the mechanisms of the cell cycle and on malignant transformation.