SPARC, a counteradhesive matricellular protein, inhibits endothelial cell adhesion and proliferation, but the pathways through which these activities are blocked are not known. In this study, we used inhibitors of major signaling proteins to identify mediators through which SPARC exerts its counteradhesive and antiproliferative functions. Pretreatments with the general protein tyrosine kinase (PTK) inhibitors, herbimycin A and genistein, protected against the inhibitory effect of SPARC on bovine aortic endothelial (BAE) cell spreading by more than 60%. Similar pretreatments with PTK inhibitors significantly blocked the diminishment of focal adhesions by SPARC in confluent BAE cell monolayers, as determined by the formation of actin stress-fibers and the distribution of vinculin in focal adhesion plaques. Inhibition of endothelial cell cycle progression by SPARC and a calcium-binding SPARC peptide, however, was not affected by PTK inhibitors. Inhibition of DNA synthesis by SPARC was not reversed by inhibitors of the activity of protein kinase C (PKC), or of cAMP-dependent protein kinase (PKA), but was sensitive to pertussis (and to a lesser extent, cholera) toxin. The counteradhesive effect of SPARC on endothelial cells is, therefore, mediated through a tyrosine phosphorylation-dependent pathway, whereas its antiproliferative function is dependent, in part, on signal transduction via a G protein-coupled receptor.