Globular adiponectin (gAd) induces the generation of reactive oxygen species (ROS) and nitric oxide (NO) in the murine macrophage cell line RAW 264. We investigated the role of Ca2+ in gAd-induced ROS and NO generation. Pretreatment with BAPTA-AM, a selective chelator of intracellular Ca2+ ([Ca2+](i)), partially reduced gAd-induced generation of ROS and NO in gAd-treated RAW 264 cells. The lowest [Ca2+](i) occurred 30 min after gAd treatment, after which [Ca2+](i) increased continually and exceeded the initial level. The mitochondrial Ca2+ ([Ca2+](m)) detected by Rhod-2 fluorescence started to increase at 6 h after gAd treatment. Pretreatment with a NAD(P)H oxidase inhibitor, diphenyleneiodonium, prevented the reduction of [Ca2+](i) in the early phase after gAd treatment. Calcium depletion by BAPTA-AM had no effect on the gAd-induced [Ca2+](m) oscillation. The administration of a specific calmodulin inhibitor, calmidazolium, significantly suppressed gAd-induced ROS and NO generation and NOS activity. (C) 2009 Elsevier Inc. All rights reserved.