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During acute rejection, leukocytes accumulate in the blood vessels of experimental renal allografts. About 70 % of them are activated monocytes. We investigated the expression and regulation of proteins involved in the synthesis of acetylcholine by intravascular allograft leukocytes. Kidneys were transplanted in the allogeneic DA to LEW and in the isogeneic LEW to LEW rat strain combination. Intravascular leukocytes were harvested four days after transplantation by extensively perfusing the blood vessels of the graft. Expression of the high-affinity choline transporter (CHT1) and of choline acetyltransferase (ChAT) were investigated by RT-PCR, immunoblotting and immunohistochemistry. CHT1 and ChAT expressing monocytes were identified by double-staining with the pan monocyte/macrophage marker ED1. Intravascular graft leukocytes expressed CHT1 and ChAT mRNA and protein. The expression of these genes was up-regulated during acute allograft rejection. On histological sections of renal allografts, virtually all intravascular leukocytes including ED1-positive monocytes were immunopositive for CHT1 and ChAT. In conclusion, we demonstrate for the first time that intravascular leukocytes including monocytes up-regulate CHT1 and ChAT during vigorous immune reactions in vivo. Thus, locally produced non-neuronal acetylcholine might function as an intercellular signal modulating immune functions inside blood vessels.