Nitrous oxide (N2O) oxidizes vitamin B12. disrupting deoxyribonucleic acid (DNA) synthesis. Occupational exposures to subanesthetic levels of the gas have been documented that may result in suppressed proliferative cell activities. Male CD-I mice were exposed to 0, 50, 500, and 5000 parts of N2O per million parts of air (ppm) for 6 hr/day, 5 days/week for 2 and 13 weeks. Splenic lymphocytes were assayed for responsiveness to mitogens and for the ability to produce interleukin-2 (lL-2) . Tritiated-thymidine ([3H]-TdR) uptake was measured in CD-I splenic lymphocytes cultured in a mixed-lymphocyte culture (MLC). Cytolytic cell activity was measured by 51chromium release assay. Antibody-mediated immunocompetency was determined for sheep red blood cell (SRBC)-sensitized animals by plaque-forming cell (PFC) assay and sera anti-SRBC antibody titer. Deoxyuridine suppression tests (dUdRST) were performed on bone marrow cells. Serum adrenocorticotropic hormone and corticosterone levels were determined. There was significantly decreased splenic lymphocyte uptake of [3H)-TdR by cells cultured with mitogenic substances and in MLC following 2-week animal exposures to 5000 ppm. After 13-week exposures, the animals' splenic lymphocytes showed decreased [3H]-TdR uptake following low N20 dosing and nonsignificantly increased responsiveness at the higher gas exposures in both the blastogenic and MLC assays. Compared to control animals, the 5000- ppm-exposure group had significantly depressed PFC activity and circulating anti-SRBC immunoglobulin M levels following 13-week gas exposures, and all three subchronic exposure groups demonstrated both decreased liver weights and leukopenia. Bone marrow activity at these dosing levels was dose-responsively depressed following subchronic gas exposures.No hormonal effect appears to be attributable to N20 exposure.