C3H/HeJ mice received multiple minor histoincompatible skin grafts (B10.BR) after portal or lateral tail vein injection of irradiated B10.BR spleen cells. Some mice were additionally injected with a competitive inhibitor of nitric oxide synthesis (NG-monomethyl-L-arginine [NMMA]). Skin graft survival was extended following portal venous immunization, and further enhanced by NMMA. Both treatments produced decreased production of nitrate/nitrite in vivo, and were associated with enhanced expression of mRNA in vivo for type 2 cytokines (interleukins 4 and 10), as well as increased synthesis of the latter on restimulation in vitro. Inducible nitric oxide synthase gene expression was up-regulated both in the local mucosal immune system (intraepithelial lymphocytes) and systemically (spleen) following antigen challenge by the portal vein or by gavage, with or without additional NMMA treatment. In contrast, when we studied a possible alternate in vivo source of nitric oxide production, we found that endothelial cell nitric oxide synthase expression was reproducibly up-regulated only in splenic tissue after combined oral (or portal) immunization and NMMA, and was not up-regulated in tissues local to the site of injection (intraepithelial lymphocytes).