In this study, we examine the effects of tissue inhibitor of metalloproteinases-2 (TIMP-2) on the phosphorylation status of specific phosphotyrosine residues on the vascular endothelial cell growth factor receptor-2 (VEGFR-2) cytoplasmic tail and examine the effects on associated downstream signaling pathways. To focus on metalloproteinase-independent mechanisms, we used the TIMP-2 analog known as Ala+ TIMP-2 that is deficient in matrix metalloproteinase-inhibitory activity. Our experiments are designed to compare the effects of VEGF-A stimulation with or without Ala+ TIMP-2 pretreatment, as well as basal responses in human microvascular endothelial cells. Our results show that Ala+ TIMP-2 selectively alters the phosphorylation pattern of VEGFR-2 after VEGF-A stimulation and disrupts the downstream activation of PLC-γ, Ca+ 2 flux, Akt, and eNOS, as well as decreasing cGMP levels. Moreover, we observed an Ala+ TIMP-2-induced reduction in cGMP levels typically elevated by exogenous NO donors implicating Ala+ TIMP-2 in the direct activation of an isobutylmethylxanthine (IBMX)-sensitive cGMP phosphodiesterase activity. TIMP-2 suppression of endothelial mitogenesis and angiogenesis involves at least two mechanisms, one mediated by protein tyrosine phosphatase inhibition of VEGFR-2 activation as well as downstream signaling and a second mechanism involving direct activation of an IBMX-sensitive phosphodiesterase activity.