Although the mitogen-activated protein kinase (MAPK) pathway has been extensively investigated, numerous events remain unclear. In the present study, we examined mitogen-activated protein kinase kinase (MEK) expression from interphase to mitosis. Following nocodazole treatment, COS7 cells gradually became round as early as 4 h after treatment. Cyclin B1 expression gradually increased from 4 to 24 h in the presence of nocodazole. When cells were treated with nocodazole for 4 h, the level of epidermal growth factor (EGF)-mediated MEK phosphorylation did not significantly change between nocodazole-untreated and -treated (4 h) cells (P>0.05). However, EGF-mediated MEK phosphorylation was significantly inhibited upon treatment with nocodazole for 8 and 24 h compared to nocodazole-untreated cells (P<0.05). MEK phosphorylation levels were comparable between 1, 5, 10 and 50 ng/ml EGF treatments. Phorbol 12-myristic 13-acetate (PMA) did not activate MEK in mitotic cells. Following treatment of COS7 cells at the interphase with AG1478 or U0126, MEK phosphorylation was blocked. In addition, the investigation of the expression of proteins downstream of MEK demonstrated that EGF does not significantly affect the phosphorylation level of extracellular-signal-regulated kinase (ERK), ribosomal protein S6 kinase (RSK) and Elk in mitotic cells (P>0.05). The results showed that MEK expression is gradually inhibited from cell interphase to mitosis, and that MEK downstream signaling is affected by this inhibition, which probably reflects the requirements of cell physiology during mitosis.