Endothelial cells in solid tumors play an important role in tumor growth, invasion and metastasis through angiogenesis. We have recently cloned two tumor vascular antigens from isolated rat tumor vascular endothelial cells (TEC). One is CD44, a family of cell surface proteins implicated in adhesion interactions and tumor metastasis. The other is OTS-8, a marker for osteoblast into osteocyte transition and type I alveolar epithelial cells termed as E11 antigen and RTI40, respectively. To test for a possible interaction between the two antigens on endothelial cells in tumor angiogenesis, we examined in vivo association of CD44 with OTS-8 using lysates of isolated rat TEC and COS-7 cells cotransfected with CD44 and OTS-8 expression plasmids. The association was detected by direct co-immunoprecipitation of the two types of cells lysed with digitonin, whereas the detection was lost when lysed with Nonidet P-40. To confirm this association, intact COS-7 cells cotransfected were reacted with homobifunctional N-hydroxysuccinimide ester crosslinking reagents. Immunoblot analysis showed a crosslinked CD44/OTS-8 protein complex of 120 kDa, suggesting the proximity of the two proteins. These findings provide evidence of a weak physical association between CD44 and OTS-8 in TEC, and suggest that OTS-8 may alter the mode of endothelial cell growth and/or migration induced by CD44 in tumor angiogenesis.