Abstract Epipolythiodioxopiperazines were tested for their immunoregulatory activity in vitro. Using the macrophage adherence test as a measure of inhibition of phagocytosis, their effect on stimulator cells in mixed lymphocyte cultures and their ability to inhibit mitogen stimulation of T lymphocytes, a hierarchy of activity was observed, with sporidesmin being the most active, followed by gliotoxin and 1,4-dimethyl-3,6-epidithio-2,5-dioxopiperazine. Derivatives of gliotoxin such as dehydro-, trisulfide and tetrasulfide gliotoxin have activities comparable to gliotoxin. The dimethylthioether derivative of gliotoxin was devoid of activity. The presence of reducing agents abrogated the activity of epi-polythiodioxopiperazines. This suggests that the bridged disulfide moiety is the single most important chemical entity for their activity. The differential activities of the active compounds may be attributable to their variations in lipophilic properties.