The addition of Ca2+-antagonizers (La2+), Ca2+-ionophores (A23187) and Ca2+-complexing agents (EGTA) inhibited the formation of xylanase activity in resting mycelia of Trichoderma reesei. The inhibition by the ionophore was reversed by the addition of Ca2+ ions. A similar inhibitory effect was obtained by the addition of the calmodulin inhibitors, trifluoroperazine, chlorpromazine and quinacrine, hence suggesting that the observed effect of Ca2+ on xylanase formation occurred via calmodulin. The inhibition of xylanase formation by trifluoroperazine was accompanied by an inhibition of formation of the xyn2 transcript, and of the hph (hygromycin B-phosphotransferase-encoding) gene when fused downstream of the 5'-regulatory signals of the T. reesei xyn2 gene, indicating that calmodulin is required for xyn2 induction. At trifluoroperazine concentrations, which inhibited extracellular xylanase formation only slightly (about 30%), the cell-free extracts exhibited slightly increased xylanase activities. Subcellular fractionation showed that in these mycelia, the XYN II protein was distributed over a range of light vesicular fractions. This accumulated XYN II protein had the same Mr as the secreted, extracellular enzyme, indicating that it had already passed Golgi-located preprotein processing. Trifluoroperazine also specifically interfered with the endogenous, Ca2+-dependent phosphorylation of a 20-kDa protein, which was predominantly observed in cell-free extracts from mycelia growing on xylan. From these data, we conclude that calmodulin is required for xylanase II formation by T. reesei both at a transcriptional level as well as at a post-Golgi step of the secretory pathway. We also suggest that at least one of these two steps may be mediated via Ca2+-calmodulin-dependent phosphorylation.