In a previous study we demonstrated that the alternatively spliced region of tenascin-C, TNfnA-D, bound with high affinity to a cell surface receptor, annexin II. In the present study we demonstrate three changes in cellular activity that are produced by adding intact tenascin-C or TNfnA-D to cells, and we show that all three activities are blocked by antibodies against annexin II. 1) TNfnA-D added to confluent endothelial cells induced loss of focal adhesions. 2) TNfnA-D produced a mitogenic response of confluent, growth-arrested endothelial cells in 1% serum. TNfnA-D stimulated mitogenesis only when it was added to cells before or during exposure to other mitogens, such as basic fibroblast growth factor or serum. Thus the effect of TNfnA-D seems to be to facilitate the subsequent response to growth factors. 3) TNfnA-D enhanced cell migration in a cell culture wound assay. Antibodies to annexin II blocked all three cellular responses to TNfnA-D. These data show that annexin II receptors on endothelial cells mediate several cell regulatory functions attributed to tenascin-C, potentially through modulation of intracellular signalling pathways.