CD44 is a widely expressed, plasma membrane protein. Many molecular forms of CD44 are possible as it is encoded by a gene with multiple exons that can be alternatively spliced and its deduced protein sequence contains numerous glycosylation sites. Through its role as an adhesion molecule, CD44 is involved in many and diverse biological processes, including angiogenesis, lymphogenesis, wound healing, inflammation, and cancer metastasis. We have developed a new panel of rat monoclonal antibodies (MAbs) to murine CD44 by immunization with mouse lung endothelial cells (LEII cells). The antibodies were characterized using immunoprecipitation, mass spectrometry, competition binding, and cross Western blot experiments with MAb 133-13A, which recognizes CD44 expressed on tumor cells. The new MAbs recognize three distinct epitope groups. MAbs 531-2A and 531-32A compete for binding with the MAb 133-13A that was described previously. MAb 531-30A identifies a CD44 epitope found on low molecular weight forms expressed in vivo, while MAb 531-22A appears to recognize an epitope specific for endothelial cells. This novel panel of anti-CD44 antibodies has potential for investigating the role of CD44 in disease pathogenesis models in the mouse. They may be particularly useful for examining the role of endothelial cells in these models.