We have been searching for antibodies reactive with rat endothelial cells. Two monoclonal antibodies (mAb), named RECA-1 and RECA-2 were produced and tested in immunoperoxidase staining on frozen sections of various rat tissues. Staining patterns were compared to those obtained with the mAbs OX-2, OX-26, OX-43, and the polyclonal antibody to von Willebrand Factor (vWF), which all have been described to react with rat endothelial cells. The RECA-2 mAb showed staining patterns similar to those obtained with OX-2. RECA-1 showed to be the only antibody reactive with all vascular endothelium in the tested tissues. In addition, RECA-1 was endothelial cell-specific whereas all other antibodies crossreacted with one or more other cell types. No reactivity of RECA-1 was found in various tested species other than rat. The RECA-1 antibody was successfully applied in staining of paraformaldehyde fixed, plastic embedded tissue material. Immunofluorescence staining of viable endothelial cells demonstrated that RECA-1 recognizes a cell surface antigen. This was supported by intravenous injection of RECA-1, which showed the antibody to localize along the endothelium lining the vasculature in various organs tested. No reactivity of the antibody was seen when applied in immunoblotting of PAGE-run lysates from endothelial cell cultures and stromal cell preparations. We believe RECA-1 to be a promising antibody for rat endothelial cell studies, and in particular for further defining nature and function of endothelial cell-specific antigens.