Tissue transglutaminase (tTgase) catalyzes the posttranslational modification of proteins by forming Ca2(+)-dependent intermolecular epsilon (gamma-glutamyl) lysine crosslinks; however, its physiological function is unclear despite increasing evidence for its involvement in the extracellular environment. To define where the enzyme is active and characterizes targets of crosslinking we have modulated tTgase expression in stably transformed Swiss 3T3 cell lines, generated by transfecting tTgase cDNA under the control of a tetracycline-regulated inducible promoter. Induced expression of tTgase enabled the detection of two pools of transglutaminase antigen, one intracellular and the other extracellular, which has a cellular distribution comparable to fibronectin. Incubation of cells with the fluorescent tTgase substrate fluorescein cadaverine indicated incorporation only in the extracellular matrix of healthy cells even though the amine was freely permeable to cells. Incorporation paralleled the deposition of fibronectin during fibril assembly when monitored by immunofluorescence. Fibronectin polymerization was confirmed by Western blotting. Cell surface-related tTgase was further demonstrated by preincubation of cells with tTgase antibody which led to inhibition of activity and cell attachment. Activation of the intracellular tTgase by increasing cytosolic Ca2+ using ionomycin resulted in cell death accompanied by extensive crosslinking in the cytoplasm, nucleus, and cell substratum contacts of induced cells. These dead cells were not typical of those undergoing apoptosis or necrosis since they remained adherent, preserved their microtubule network, and showed little DNA fragmentation. Modulation of expression of tTgase has indicated a possible physiological function for the enzyme in cell attachment, the crosslinking of fibronectin during fibril assembly, and the maintenance of cellular integrity in a novel form of cell death.