Big MAP kinase 1 (BMK1), also known as ERK5, is a mitogen-activated protein (MAP) kinase member whose biological role is largely undefined. We have shown previously that the activity of BMK1 in rat smooth muscle cells is up-regulated by oxidants. Here, we describe a constitutively active form of the MAP kinase kinase, MEK5(D), which selectively activates BMK1 but not other MAP kinases in vivo. Through utilization of MEK5(D), we have determined that a member of the MEF2 transcription factor family, MEF2C, is a protein substrate of BMK1. BMK1 dramatically enhances the transactivation activity of MEF2C by phosphorylating a serine residue at amino acid position 387 in this transcription factor. Serum is also a potent stimulator of BMK1-induced MEF2C phosphorylation, since a dominant-negative form of BMK1 specifically inhibits serum-induced activation of MEF2C. One consequence of MEF2C activation is increased transcription of the c-jun gene. Taken together, these results strongly suggest that in some cell types the MEK5/BMK1 MAP kinase signaling pathway regulates serum-induced early gene expression through the transcription factor MEF2C.