Two-hybrid screening of a tobacco BY-2 cell suspension cDNA library using the p43(Ntf6) mitogen-activated protein (MAP) kinase as bait resulted in the isolation of a cDNA encoding a protein with features characteristic of a MAP kinase kinase (MEK), which has been called NtMEK1. Two-hybrid interaction analysis and pull-down experiments showed a physical interaction between NtMEK1 and the tobacco MAP kinases p43(Ntf6) and p45(Ntf4), but not p43(Ntf3). In kinase assays NtMEK1 preferentially phosphorylated p43(Ntf6). Functional studies in yeast showed that p43(Ntf6) could complement the yeast MAP kinase mutant mpk1 when co-expressed with NtMEK1, and that this complementation depended on the kinase activity of p43(Ntf6). Expression analysis showed that the NtMEK1 and ntf6 genes are co-expressed both in plant tissues and following the induction of cell division in leaf pieces. These data suggest that NtMEK1 is an MEK for the p43(Ntf6) MAP kinase.