The regulation of the MAPKs, Erk₁ and Erk₂, and the MAPK kinase, Mek, were examined in the Y1 mouse adrenocortical tumor cell line and in the protein kinase A-defective mutant, Kin-8. ACTH and basic fibroblast growth factor each increased Mek phosphorylation and stimulated Mek activity in both cell lines and also activated the Erks at concentrations that paralleled their effects on Mek. The specific Mek inhibitor, PD98059, blocked the activation of the Erks by ACTH and basic fibroblast growth factor, indicating that Mek is the upstream activator of Erk. PD98059 did not block the phosphorylation of Mek, as might have been expected from previous studies; instead PD98059 inhibited the activity of the activated enzyme. In ACTH-stimulated, mutant Kin-8 cells, PD98059 paradoxically increased the amount of phosphorylated Mek, while preventing the activation of Erk. These results are interpreted as reflecting the loss of a protein kinase A-mediated inhibitory influence on Mek phosphorylation and activation.