Autotaxin or NPP2 (nucleotide pyrophosphatase/phosphodiesterase 2) is a secreted lysophospholipase-D that promotes metastasis and tumor growth by its ability to generate lysophosphatidic acid. Considerable evidence suggests that inhibitors of NPP2 can be used as a novel therapy for the treatment of cancer. Although most attention is currently directed toward the development of inhibitors of the catalytic site, we have explored whether NPP2 can also be targeted through its non-catalytic nuclease-like domain. We demonstrate here that the catalytic and nuclease-like domains are covalently linked by an essential disulfide bridge between Cys413 and Cys805. Within the nuclease-like domain, residues 829-850 are involved in the secretion of NPP2, and Lys852 is required for the expression of catalytic activity. These data show that the nuclease-like domain is crucial for catalysis by NPP2 and is a possible target to generate inhibitors.