Inhibition of eosinophil apoptosis by exposure to interleukin-5 (IL-5) is associated with the development of tissue eosinophilia and may contribute to the inflammation characteristic of asthma. Analysis of the signaling events associated with this process has been hampered by the inability to efficiently manipulate eosinophils by the introduction of active or inhibitory effector molecules. Evidence is provided, using a dominant-negative N17 H-Ras protein (dn-H-Ras) and MEK inhibitor U0126, that activation of the Ras-Raf-MEK-ERK pathway plays a determining role in the prolongation of eosinophil survival by IL-5. For these studies, a small region of the human immunodeficiency virus Tat protein, a protein transduction domain known to enter mammalian cells efficiently, was fused to the N-terminus of dn-H-Ras. The Tat-dn-H-Ras protein generated from this construct transduced isolated human blood eosinophils at more than 95% efficiency. When Tat-dn-H-Ras-transduced eosinophils were treated with IL-5, they exhibited a time- and dosage-dependent reduction in extracellular regulated kinase 1 and 2 activation and an inhibition of p90 Rsk1 phosphorylation and IL-5-mediated eosinophil survival in vitro. In contrast, Tat-dn-H-Ras did not inhibit CD11b up-regulation or STAT5 tyrosine phosphorylation. These data demonstrate that Tat dominant-negative protein transduction can serve as an important and novel tool in studying primary myeloid cell signal transduction in primary leukocytes and can implicate the Ras-Raf-MEK-ERK pathway in IL-5-initiated eosinophil survival.