Previously, we suppressed the expression of genes encoding isozymes of lignin peroxidase (LiP) and manganese peroxidase (MnP) using a calmodulin (CaM) inhibitor, W7, in the white-rot fungus Phanerochaete chrysosporium; this suggested that CaM positively regulates their expression. Here, we studied the role of CaM in another white-rot fungus, Pleurotus ostreatus, which produces MnP and versatile peroxidase (VP), but not LiP. W7 upregulated Mn(2+)-dependent oxidation of guaiacol, suggesting that CaM negatively regulates the production of the enzymes. Suppression of CaM in P. ostreatus using RNAi also led to upregulation of enzyme activity, whereas overexpression of CaM in P. ostreatus caused downregulation. Real-time RT-PCR showed that MnP1-6 and VP3 levels in the CaM-knockdown strain were higher than those in the wild-type strain, while MnP-5 and -6 and VP1 and 2 levels in the CaM-overexpressing strain were lower than in the wild type. Moreover, we also found that another ligninolytic enzyme, laccase, which is not produced by P. chrysosporium, was negatively regulated by CaM in P. ostreatus similar to MnP and VP. Although overexpression of CaM did not reduce the ability of P. ostreatus to digest beech wood powder, the percentage of lignin remaining in the digest was slightly higher than in the wild-type strain digest.