Evaluation of the activation state of protein kinase C (PKC) isozymes relies on analysis of subcellular translocation. A monoclonal antibody, 14E6, specific for the activated conformation of εPKC, was raised using the first variable (V1) domain of εPKC as the immunogen. 14E6 binding is specific for εPKC and is greatly increased in the presence of PKC activators. Immunofluorescence staining by 14E6 of neonatal rat primary cardiac myocytes and the NG108-15 neuroblastoma glioma cell line, N.G108-15/D2, increases rapidly following cell activation and is localized to new subcellular sites. However, staining of translocated εPKC with 14E6 is transient, and the epitope disappears 30 min after activation of NG-108/15 cells by a D2 receptor agonist. In contrast, subcellular localization associated with activation, as determined by commercially available polyclonal antibodies, persists for at least 30 min. In vitro, εRACK, the receptor for activated εPKC, inhibits 14E6 binding to εPKC, suggesting that the 14E6 epitope is lost or hidden when active εPKC binds to its RACK. Therefore, the 14E6 antibody appears to identify a transient state of activated but non-anchored εPKC. Moreover, binding of 14E6 to εPKC only after activation suggests that lipid-dependent conformational changes associated with εPKC activation precede binding of the activated isozyme to its specific RACK, εRACK. Further, monoclonal antibody 14E6 should be a powerful tool to study the pathways that control rapid translocation of εPKC from cytosolic to membrane localization on activation.