A total of 13 rabbit endothelial cell lines were examined for the presence, distribution, and localization of the thrombomodulin (TM) antigen. Three endothelial cell lines were derived from aorta, 5 lines from brain microvessels, and 5 lines from fat microvessels. Immunofluorescence demonstrated the presence of the TM antigen in all cell endothelial lines examined, at all passage levels, and at all stages of growth i.e., log phase, confluent, postconfluent stationary. The percentage of positive cells in a given culture and the distribution of the TM antigen within the cells, as well as on their surfaces, was a cell line characteristic. The intercellular variation in the expression of the TM antigen was insignificant in some cell lines but was striking in others. These characteristics were clonable. Immunofluorescence indicated and immunoperoxidase electron microscopy confirmed the localization of the TM antigen to the outer cell surface and in the Golgi-endoplasmic reticulum (GERL) complex.