Exposure of neutrophils to a variety of agonists including chemoattractant peptides and cytokines induces degranulation and activation of the oxidative burst which are required for bacterial killing. The signaling pathways regulating these important functions are incompletely characterized. Mitogen-activated protein (MAP) kinases, which include the extracellular signal-regulated kinases (ERKs), are activated rapidly in neutrophils, suggesting that they may regulate cell activation. We found that neutrophils express two isoforms of MAP/ERK kinase (MEK), mixed-function kinases that are responsible for phosphorylation and activation of ERK. Like MEK-1, MEK-2 was found to reside in the cytosol both before and after stimulation. Studies were undertaken to define the relative abundance and functional contribution of MEK-1 and MEK-2 in neutrophils and to characterize the signaling pathways leading to their activation. Although the abundance of the two isoforms was similar, the activity of MEK-2 was at least 3-fold greater than that of MEK-1. A rise in cytosolic [Ca2+] was insufficient for MEK stimulation, and blunting the [Ca2+] change with intracellular chelators failed to prevent receptor-mediated activation of either isoform, implying that cytosolic Ca2+ transients are not necessary. In contrast, both MEK-1 and MEK-2 were activated by exposure of cells to protein kinase C (PKC) agonists. Conversely, PKC antagonists inhibited the chemotactic stimulation of both isoforms, suggesting that PKC was required for their activation. Despite these similarities, clear differences were also found in the pathways leading to activation of the MEK isoforms. In particular, MEK-2 was considerably more sensitive than MEK-1 to the phosphatidylinositol 3-kinase inhibitor wortmannin. Phosphorylation and activation of ERK-1 and ERK-2 were also reduced by this inhibitor. In summary, MEK-2 is stimulated in formyl-methionyl-leucyl-phenylalanine-treated neutrophils, where it appears to be functionally the predominant isoform. The time course and inhibitor sensitivity of MEK-2 activation parallel those of several components of the microbicidal response, suggesting a signaling role of the MEK-ERK pathway.