Aortic smooth muscle cell release of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) has been implicated in aortic aneurysm pathogenesis, but proximal modulation of release is poorly understood. Extracellular nucleotides regulate vascular smooth muscle cell metabolism in response to physiochemical stresses, but nucleotide modulation of MMP and/or TIMP release has not been reported. We hypothesized that nucleotides modulate MMP-2 and TIMP-2 release from human aortic smooth muscle cells (HASMCs) via distinct purinergic receptors and signaling pathways. We exposed HASMCs to exogenous ATP and other nucleotides with and without interleukin 1-beta (IL-1). HASMCs were pretreated in some experiments with apyrase, which degrades ATP, and inhibitors of ERK1/ERK2, JNK, and p38MAPK. MMP-2 and TIMP-2 released into supernatant were assessed by ELISA and Western blotting. ATP, adenosine, and UTP significantly stimulated MMP-2 release in the presence of IL-1 (300nM ATP: 181±22%, p=0.003; 30µM adenosine: 244±150%, p=0.001; 200µM UTP 153±40%, p=0.015; vs. 100% constitutive). ATP also stimulated MMP-2 release in the absence of IL-1 (100µM ATP: 148±38% vs. 100% constitutive). Apyrase significantly reduced ATP-stimulated MMP-2 release (apyrase+500nM ATP: 59±3% vs. 124±7% with 500nM ATP). Rank-order agonist potency for MMP-2 release was consistent with ATP activation of P₂Y₁ and P₂Y₂ receptors. ATP induced phosphorylation of intracellular JNK and inhibition of the JNK pathway blocked ATP-stimulated MMP-2 release indicating signaling via this pathway. Nucleotides are thus novel stimulants of MMP-2 release from human aortic smooth muscle cells and may provide a mechanistic link between physiochemical stress in the aorta and aneurysms, especially in the context of inflammation.