Extracellular pyrophosphate (PPi) is a potent suppressor of physiological calcification in bone and pathological calcification in blood vessels. Ecto-nucleotide pyrophosphatase/ phosphodiesterases (eNPPs) generate PPi via the hydrolysis of ATP released into extracellular compartments by poorly understood mechanisms. Here we report that cultured vascular smooth muscle cells (VSMC) from rat aorta generate extracellular PPi via an autocrine mechanism that involves ATP release tightly coupled to eNPP activity. The nucleotide analog -methylene ATP (MeATP or AMPPCP) was used to selectively suppress ATP metabolism by eNPPs but not the CD39-type ecto-ATPases. In the absence of MeATP, VSMC generated extracellular PPi to accumulate >600 nM within 2 hr while steadily maintaining extracellular ATP at 1 nM. Conversely, the presence of MeATP completely suppressed PPi accumulation while increasing ATP accumulation. Probenencid (PB), which inhibits PPi efflux dependent on ANK, a putative PPi transporter or transport regulator, reduced extracellular PPi accumulation by ~2-fold. This indicates that autocrine ATP release coupled to eNPP activity comprises >50% of the extracellular PPi-generating capacity of VSMC. The accumulation of extracellular PPi and ATP was markedly attenuated by reduced temperature but was insensitive to brefeldin-A which suppresses constitutive exocytosis of Golgi-derived secretory vesicles. The magnitude of extracellular PPi accumulation in VSMC cultures increased with time post-plating suggesting that ATP release coupled to PPi generation is up-regulated as cultured VSMC undergo contact-inhibition of proliferation or deposit extracellular matrix.