MEK partner 1 (MP1) and P14 are small proteins that modulate the Raf-MKK1/2-ERK1/2 pathway. To examine the biochemical basis for their function a fluorescent form of MP1 was prepared by labeling Cys-74 with fluorescein. Using this protein it was shown that MP1 binds unactivated ERK1, ERK2 and a constitutively active form of MKK1 (MKK1G7B) with dissociation constants of 9.7+/-1.6, 3.3+/-0.6 and 2.2+/-0.5 microM, respectively. MP1 inhibits the ability of activated ERK2 to phosphorylate the transcription factor Ets-1. Both MP1 and the MP1*P14 complex inhibit the ability of activated ERK2 to phosphorylate MKK1G7B, thus impeding feedback inhibition. In contrast, MP1 and the P14*MP1 complex enhance the ability of MKK1G7B to phosphorylate ERK2, when ERK2 is present at a low concentration, but not when it is present at a high concentration. Thus, MP1 and the MP1*P14 complex have the potential to differentially modulate activating and inhibiting signals in the Raf-MKK1/2-ERK1/2 pathway.