A series of studies was performed in tumor bearing mice to evaluate the impact of glutathione (GSH) depletion by L-buthionine sulfoximine (L-BSO). L-BSO doses of 50 or 500 mg/kg were used alone or with one of 4 sulfhydryl-dependent anticancer agents (SHDAA). When L-BSO was administered to tumor-bearing mice, Colon 38 cells were significantly depleted of GSH content, but this did not occur with P388 cells or MOPC-315 cells in vivo. GSH levels in these ascites tumors declined significantly without L-BSO treatment as the tumor rapidly grew in the IP space. SHDAAs, including doxorubicin (DOX), cyclophosphamide (CTX), carmustine (BCNU) and melphalan (L-PAM) were then combined with L-BSO in mice bearing P388, MOPC-315 or colon 38 tumors. There was no consistent enhancement of antitumor efficacy using a treatment interval of 24 hrs (L-BSO given first). In contrast, there was some evidence of significantly enhanced SHDAA toxicity with L-BSO. Further studies should evaluate different dosing intervals to take advantage of the slower rate of GSH replenishment observed in normal tissues compared to solid tumor cells (Colon 38) in vivo. In addition, significant reductions for any SHDAA combined with L-BSO are indicated in any such trial.