Thioredoxin is a cytosolic, redox-active protein that is secreted from many cells and has several extracellular functions. In activated lymphocytes, the pathway of secretion does not involve the Golgi apparatus. Levels of extracellular thioredoxin are decreased by the antioxidant N-acetylcysteine. Hence, the secretion of thioredoxin could be altered by the redox status of the cell or the protein. To study thioredoxin mutants, we characterized the secretion of human thioredoxin from Chinese hamster ovary cells. Secretion of human thioredoxin is unaffected by brefeldin A, slow but efficient, and sensitive to low temperature and factors in serum. We demonstrate that N-acetylcysteine reduces the cellular level of thioredoxin but not the proportion secreted; thus, this chemical does not block the non-classical pathway for thioredoxin secretion. Furthermore, we find that mutations in either the active site or the dimerization site of thioredoxin do not alter its secretion. Thus, the non-classical secretion of thioredoxin is not dependent on the redox status of either the cell or the protein.