The c-jun N terminal kinase (JNK) signaling pathway mediates interleukin-1 (IL-1)-induced apoptosis in insulin-secreting cells, a mechanism relevant to the destruction of pancreatic -cells in type 1 and 2 diabetes. However, the mechanisms that contribute to IL-1 activation of JNK in -cells are largely unknown. In this study we investigated if Ca²⁺ plays a role for IL-1-induced JNK activation. In insulin-secreting rat INS-1 cells cultured in the presence of 11 mM glucose, combined pharmacological blockade of L- and T-type Ca²⁺ channels suppressed IL-1-induced in vitro phosphorylation of the JNK substrate c-jun and reduced IL-1-stimulated activation of JNK1/2 as assessed by immuno blotting. Inhibition of IL-1-induced in vitro kinase activity toward c-jun following collective L- and T-type Ca²⁺ channel blockade was confirmed in primary rat and ob/ob mouse islets and in mouse TC3 cells. Ca²⁺ influx specifically via L-type, but not T-type channels contributed to IL-1 activation of JNK. Activation of p38 and extracellular signal-regulated kinase in response to IL-1 was also dependent on L-type Ca²⁺ influx. Membrane depolarization by KCl, exposure to high glucose, treatment with Ca²⁺ ionophore A23187 or exposure to thapsigargin, an inhibitor of sarco(endo)plasmic reticulum Ca²⁺-ATPase, all caused an amplification of IL-1-induced JNK activation in INS-1 cells. Finally, a chelator of intracellular free Ca²⁺ (BAPTA-AM), an inhibitor of calmodulin (W7), and inhibitors of Ca²⁺/calmodulin-dependent kinase (KN62 and KN93) partially reduced IL-1-stimulated c-jun phosphorylation in INS-1 or TC3 cells. Our data suggest that Ca²⁺ plays a permissive role in IL-1 activation of the JNK signaling pathway in insulin-secreting cells.