A persisting paradox in cytoskeletal regulation of cell motility is the loss of the actin filament fragmenting protein, gelsolin, in transformed epithelial cells that have gained the ability to migrate. Either actin filament severing does not occur during motility of carcinoma cells or a novel fragmentation protein is expressed during transformation. Using an antibody specific for severin, the Mr 40,000 actin filament severing protein from Dictyostelium discoideum amoebae, we have identified a mammalian form of severin in murine LL/2 carcinoma cells lacking gelsolin. Mammalian severin (M-severin) isolated from LL/2-derived Lewis lung carcinoma tumors severed F-actin in a calcium-dependent manner, mimicking the function of Dictyostelium severin. M-severin preferentially localized to the cleavage furrow of dividing LL/2 cells and to the actin-rich cortex of migratory LL/2 cells, known sites of active actin cytoskeleton rearrangement. The mammalian severing protein was fully expressed in transformed LL/2 epithelial cells but went undetected in normal mouse muscle, liver, spleen, or kidney. Normal mouse lung tissue contained minute amounts of M-severin, attributed to motile cells in pulmonary connective tissue. In striking contrast to M-severin, gelsolin was highly expressed in normal lung but disappeared in transformed LL/2 carcinoma cells. Based on prior observations of a functional role for actin filament fragmentation in cell migration, the simultaneous induction of M-severin and loss of gelsolin during epithelial transformation suggests that replacement of gelsolin by M-severin may function to achieve actin filament rearrangements necessary for active cell migration in invasive or metastatic carcinoma. Induction of M-severin in an invasive tumor was directly observed in human colon adenocarcinoma by cytoimmunohistochemistry with antibodies directed against severin isolated from both Dictyostelium amoebae and Lewis lung carcinoma cells. Because normal colon epithelium from the same patient did not express M-severin, it may serve as a sensitive marker for detection and staging of epithelial tumors.