Alternative splicing of the caldesmon gene results in high (h-caldesmon) and low (l-caldesmon) molecular weight isoforms of the cytoskeleton-associated protein caldesmon. h-Caldesmon is predominantly expressed not only in smooth-muscle cells but also in pericryptal fibroblasts in colon. l-Caldesmon is widely expressed and localized in podosomes/invadopodia. Studies with transformed and cancer cell lines suggest that a reduction in l-caldesmon facilitates podosome/invadopodia formation and metastasis. We investigated caldesmon isoforms in colon adenocarcinoma and their lymph node metastases using immunohistochemistry. Caldesmon immunoreactivity of colon adenocarcinoma primary tumors and lymph node metastases was very similar. l-Caldesmon immunoreactivity of cancer epithelial cells in primary tumors and lymph node metastases varied ranging from reduced to stronger as compared to immunoreactivity of normal areas. The variation did not show a consistent relation to the tumor center or invasive margin. While h-caldesmon immunoreactivity of pericryptal fibroblasts and blood vessels in the stroma of primary tumors and lymph node metastases was markedly reduced, cancer-associated fibroblasts and blood vessels in the stroma were strongly immunoreactive for l-caldesmon. Our results show a differential behavior of h- and l-caldesmon isoforms in epithelium and stroma of colon adenocarcinoma and lymph node metastases.