Nanosecond pulsed electric fields (nsPEFs) are increasingly being recognized as a potential tool for use in the life sciences. Exposure of human cells to nsPEFs elicits the formation of small membrane pores, intracellular Ca2+ mobilization, signaling pathway activation, and apoptosis. Here we report the activation of AMP-activated protein kinase (AMPK) by nsPEFs. AMPK activation is generally achieved by the phosphorylation of AMPK in response to changes in cellular energy status and is mediated by two protein kinases, LKB1 and CaMKK. Exposure to nsPEFs rapidly induced phosphorylation of AMPK and its downstream target ACC in both LKB1-proficient and LKB1-deficient cells. In LKB1-deficient cells, AMPK activation by nsPEFs was mediated by CaMKK and required extracellular Ca2+, which suggested the occurrence of Ca2+ mobilization and its participation in AMPK activation by nsPEFs. Our results provide experimental evidence for a direct link between activated cellular signaling and Ca2+ mobilization in nsPEF-exposed cells. (C) 2012 Elsevier Inc. All rights reserved.