Endogenous mammalian lectin-like sugar-binding molecules have been previously described that have immunoregulatory properties. Further, the addition of defined simple saccharides to lymphocyte cultures has been shown to inhibit a variety of in vitro lymphocyte functions, presumably because these sugars are able to compete with the binding of endogenous lectins to critical membrane receptors. In this report, we describe the isolation and characterization of a D-mannose-containing disaccharide in human pregnancy urine that inhibits the proliferative response of human T lymphocytes. The inhibitory disaccharide was purified to homogeneity by sequential steps including affinity chromatography on immobilized concanavalin A and molecular sizing on Sephadex G-75 and then Fractogel 40S columns, with final purification on high-performance thin-layer chromatography. By mass spectrometry of the purified material as its permethylated derivative, the deduced structure of this compound was alpha-D-Manp 1-6-D-Man. To confirm that this disaccharide was in fact immunosuppressive, an identical disaccharide was prepared by sequential digestion of yeast cell wall polysaccharide. The urinary and yeast disaccharides had identical immunosuppressive properties. It has been previously reported that D-mannose is inhibitory for antigen-specific proliferative assays in the range of 10-50 mM. The purified alpha-D-Manp 1-6-D-Man disaccharide was inhibitory at 100-fold-lower concentrations. Further, while D-mannose inhibits T cell proliferation when added at anytime up to 24 h before harvest of a 6-d lymphocyte culture, alpha-D-Manp 1-6-D-Man disaccharide was inhibitory only if added at the initiation of culture and had no inhibitory effect if added just 24 h later. These data support the concept that simple sugar compounds can exhibit marked immunoregulatory activity in vitro. The impact of these molecules on the regulation of immune responses in vivo is unknown, as is their precise mechanism of action, but structural and chemical identification should now permit a detailed analysis of these issues.