We recently reported that leukemia inhibitory factor (LIF) enhances Ca2+]ithrough an increase in L-type Ca2+current (ICa,L) in adult cardiomyocytes. The aim of this study was to investigate whether LIF activates Ca2+-dependent signaling molecules, such as calcineurin and calmodulin kinases II and IV (CaMKII and CaMKIV), and, if so, whether these Ca2+-mediated signaling events contribute to LIF-mediated cardiac hypertrophy. We first confirmed that LIF increasedICa,Land [Ca2+]iin primary cultured rat neonatal cardiomyocytes. Calcineurin, CaMKII, and CaMKIV activities increased at 2 minutes and peaked by 1.6-, 2.2-, and 2.2-fold, respectively, at 15 minutes. Nicardipine or verapamil fully inhibited these activities. Autophosphorylation of CaMKII was also observed to parallel the timing of CaMKII activity, and this phosphorylation was blocked by nicardipine, verapamil, or EGTA. LIF treatment led to a 3-fold increase in nuclear factor of activated T cell–luciferase activity. To confirm that inositol triphosphate (IP3)-induced Ca2+release from sarcoplasmic reticulum was not involved in this process, IP3content and phosphorylation of phospholipase C&ggr; were investigated. LIF did not increase IP3content or phosphorylate phospholipase C&ggr;. KN62 (an inhibitor of CaMKII and CaMKIV) attenuated c-fos, brain natriuretic peptide, &agr;-skeletal actin, and atrial natriuretic peptide expression. KN62 suppressed the LIF-induced increase in [3H]phenylalanine uptake and cell size. Cyclosporin A and FK506 slightly attenuated brain natriuretic peptide but did not affect c-fos or atrial natriuretic peptide expression. Cyclosporin A significantly reduced the LIF-induced increase in [3H]phenylalanine uptake. These findings indicated that LIF activated CaMKII, CaMKIV, and calcineurin through an increase inICa,Land [Ca2+]iand that CaMKII, CaMKIV, and calcineurin are critically involved in LIF-induced cardiac hypertrophy.