Calcium (Ca2+) is a second messenger in many physiological and phyto-pathological processes. Peroxisomes are subcellular compartments with an active oxidative and nitrosative metabolism. Previous studies have demonstrated that peroxisomal nitric oxide (NO) generation is dependent on calcium and calmodulin (CaM). We used Arabidopsis thaliana transgenic seedlings expressing cyan fluorescent protein (CFP) through the addition of peroxisomal targeting signal 1 (PTS1), which enables peroxisomes to be visualized in vivo, and also used a cell-permeable fluorescent probe for Ca2+. Analysis by confocal laser scanning microscopy (CLSM) enabled us to visualize the presence of endogenous Ca2+ in the peroxisomes of both roots and guard cells. The presence of calcium in peroxisomes and the import of CFP-PTS1 are drastically disrupted by both CaM antagonist and glutathione (GSH). Furthermore, the activity of three peroxisomal enzymes (catalase, glycolate oxidase and hydroxypyruvate reductase) containing PTS1 was clearly affected, with a decrease of between 41 and 51 %. In summary, data show that CaM/Ca2+ is strictly necessary for protein import, normal functionality of peroxisomal enzymes, including antioxidant and photorespiratory enzymes, as well as for NO production.