We have investigated the activity of the Ca(2+)-dependent apoptosis-related transglutaminase type 2 in the mnd/mnd mouse mutant. Transglutaminase activity in mnd/mnd central nervous system (CNS) tissue homogenates was identical to that of healthy animals at 3 months of age, but at 8 months it was greater in the mnd/mnd CNS by up to four times, depending on the region. Western blot analysis showed no difference in the level of immunoreactive transglutaminase type 2 in spinal cord homogenates between mnd/mnd and healthy mice. However, a greater number of acyl donor protein substrates of transglutaminase were identified in mnd/mnd tissue. N epsilon (gamma-Glutamyl)lysine cross-linked product of transglutaminase activity was localized to the soma of degenerating motor neurons in the mnd/mnd mouse spinal cord. We conclude that neurodegeneration in the mnd/mnd mouse is accompanied by activation of transglutaminase at substrate level. Possible mechanisms of activation and its implications for cellular pathology are discussed.