Methotrexate action in viable cells was monitored by registering changes in EGFP (Enhanced Green Fluorescent Protein) fluorescence intensity. Treatment with 1 microM methotrexate for 48 h of human colorectal adenocarcinoma C85 cells, stably transfected to express EGFP, caused 5-fold increase in EGFP fluorescence assayed by flow cytometry with no distinct increase in EGFP protein level. This was correlated with morphological changes, including an increase of cell granularity and cell shape flattening, as well as cell cycle G1 phase arrest revealed by DNA content analysis. Methotrexate removal allowed the morphology of the cells in culture to revert in 10 days to normal. The cells that survived methotrexate exposure were propagated as C85r cell subline and displayed kinetics of methotrexate sensitivity parallel to that of the parental C85 line. As the increase in EGFP fluorescence could also be visualized by fluorescence microscopy, this reporter system may be employed to image methotrexate action in cancer cells in living models.