Supernatants derived from unstimulated cultures of mononuclear leucocytes obtained from healthy subjects contained a factor(s) which consistently suppressed lymphocyte proliferative responses to antigens and mitogens. This factor(s) is produced by a non-adherent cell and was generated in vitro after 4 hr of culture. Cells from almost all healthy subjects produced this substance(s). Cell number and viability were not affected by it. Kinetic studies suggested that interference with antigen presentation was not the mechanism of its action. The release of this immune response suppressor factor(s) (IRSF) was not blocked by indomethacin and its biological activity was unrelated to levels of prostaglandin E2. Experiments with low-specific-activity thymidine showed that suppression was not due to release of unlabelled nucleotide. Preliminary characterization of IRSF revealed that it is heat-stable and partially dialysable through membranes with an exclusion size of 12,000 daltons. IRSF differs from previously reported soluble suppressor substances and may play a role in immunoregulation in health.