The proposed research deals with an enzymic approach to the amelioration of toxicity associated with methotrexate (MTX) therapy. A Flavobacterium sp. carboxypeptidase that hydrolyzes MTX in vitro 20 times more rapidly than 5-methyltetrahydrofolate (meFH4) is considered for its potential value as a rescue agent in the combination therapy of solid tumors with MTX. A detailed evaluation of the carboxypeptidase is the central theme of the experimental plan. It consists of in vitro and in vivo aspects of enzyme characterization, differential reactivity with MTX and meFH4, and assessment of efficacious and selective antagonism of MTX toxicity. Pharmacokinetic studies will be emphasized. The investigation of the ability of the enzyme to improve the efficacy of MTX in the therapy of a solid tumor in rats will be conducted with the Walker carcinosarcoma 256. This tumor is relatively sensitive to high-dose MTX. Experimental trials will explore a range of MTX and enzyme doses with continuous infusion of MTX and varied scheduling of carboxypeptidase administration. The possibility of problems arising from the formation of 4-amino-10-methylpteroic acid as the degradation product of MTX and from the immunogenicity of the carboxypeptidase will be investigated. A plan for the resolution of those problems is presented.