Nuclear factor-κB (NF-κB) plays an important role in inflammation, proliferation and regulation of apoptosis. The purpose of the present study on type II cells was to investigate whether Chlamydophila pneumoniae contact induces (I) a Ca²⁺ release, that (II) disrupts F-actin/β-tubulin cytoskeletal association with NF-κB/ IκBα, leading to (III) a subsequent NF-κB activation. Incubation of rat type II pneumocytes with C. pneumoniae caused an intracellular calcium release within seconds. Confocal laser scanning microscopy (CLSM) revealed that bacterial contact with cell surface leads to a disappearance of the microvilli and disturbs the co-localization between F-actin and NF-κB (p65). Using semi-quantitative CLSM, we show that at 10-30 min IκBα was decreased and p65 or p50 was simultaneously translocated from cytoplasm to the nucleus, resulting in a 19-fold and 17-fold increase versus control cells. During this time no bacteria were internalized into type II cells. The pre-treatment of cells with BAPTA-AM inhibited C. pneumoniae-mediated calcium release. BAPTA-AM or SN50 prevented the C. pneumoniae-induced changes in F-actin cytoskeleton and inhibited NF-κB activation. Paclitaxel reduced C. pneumoniae-mediated changes of β-tubulin cytoskeleton and activation of NF-κB. These results suggest that calcium-mediated cytoskeleton reorganization is involved in C. pneumoniae-induced NF-κB activation in type II cells.