The aim of this study was to examine the expression of methylthioadenosine phosphorylase (MTAP) in 21 fresh tumor samples from patients with soft tissue sarcomas (STS) and 11 human soft tissue sarcoma cell lines, and to determine if loss of expression of this enzyme was correlated with increased sensitivity to L-alanosine and/or 6-methylmercaptopurine. We used a polyclonal antibody to measure the expression of MTAP in soft tissue sarcoma cell lines and in fresh tumor samples. Transfection of the HT-1080 cell line with a plasmid containing the cDNA for the MTAP gene was also performed to generate cell lines for in vitro and in vivo comparative sensitivity studies. MTAP was not expressed in 8 of 21 fresh STS tumors. The expression of MTAP was also not detectable in 3 of the 11 soft tissue sarcoma cell lines (HT-1080, HS42, and M-9 110). These three cell lines were more sensitive to L-alanosine, a potent inhibitor of de novo AMP synthesis, and to an inhibitor of de novo purine nucleotide synthesis, 6-methylmercaptopurine riboside (MMPR). The IC50 values for L-alanosine and MMPR were >20-fold lower in MTAP-deficient cells than in MTAP-positive cells. Restoration of MTAP into HT-1080 MTAP-deficient cells also led to decreased sensitivity to L-alanosine and MMPR. An in vivo study using HT-1080 cell tumors with and without MTAP expression confirmed that tumors lacking MTAP were more sensitive to L-alanosine than tumors expressing MTAP. These results provide the basis for selective therapy using inhibitors of de novo purine nucleotide synthesis such as L-alanosine or MMPR to treat patients with STS lacking this enzyme.