The effect of transient transfection with expression vectors of Ha-ras on the promoter activity of 12-lipoxygenase in human epidermoid carcinoma A431 cells was studied. Overexpression of Ha-ras increased the promoter activity in a dose- and time-dependent manner, which correlated closely with the cellular expression of Ras protein. Promoters of different gene lengths for human 12-lipoxygenase were used to prepare the luciferase fusion vectors. Following transfection by Ha-ras for 68 h, an approx. 40-fold increase in luciferase reporter activity was observed in plasmids with the 5′-flanking region ranging from −951 to −224 bp upstream from translation starting site. There was no obvious stimulation in cells transfected with a vector-bearing promoter with a length of −100 bp. These results indicate that the promoter region ranging from −224 to −100 bp was important for the Ha-ras response. With the aid of additional 5′-deletion and site-directed mutagenesis, three Sp1 binding sequences residing at −169 to −161 bp, −158 to −150 bp and −123 to −114 bp were found to be critical for the Ha-ras response of activating the transcription of human 12-lipoxygenase gene.