In this NCI Outstanding Investigator Grant Application the principal investigator has developed the evolution of his research in the area of the cellular pharmacology of anticancer drugs emerging from a basic background in membrane transport to a more comprehensive approach to study of the interaction between anticancer drugs and the intact cell. This is an approach that distinguishes among membrane transport, drug metabolism, interactions between drugs, metabolites and intracellular targets and the role of these various interactions as determinants of drug cytotoxicity, selectivity and resistance. A major emphasis of the investigator's work has been in the area of 4-amino antifolate pharmacology, with contributions in the area of membrane transport, the nature of the interaction between methotrexate and dihydrofolate reductase in cells and, more recently, comprehensive studies to explore the polyglutamylation of methotrexate, its 7-hydroxy catabolite and aminopterin. This research has been done within the context of interactions and collaborations with faculty involved in a basic membrane biology research and research training program. This has supported basic studies in membrane transport, the application of network thermodynamic modelling to these studies and provides collaborative support for some of the proposed studies. The applicant proposes to continue extensive studies to further explore polyglutamylation of 4-amino antifolates and to assess the cellular and sub- cellular effects of monoglutamyl and polyglutamyl 4-amino antifolates on metabolism of exogenously provided and endogenous tetrahydrofolate cofactors. Other studies will explore the mechanisms of membrane transport of 4-amino folates and the natural tetrahydrofolate cofactors and how intracellular folyl or antifolyl polyglutamates affect these processes. Two new initiatives are proposed. First, studies will assess the effects of 4- amino antifolates on the functional properties of renal tubular epithelial cell monolayers to explore on a cellular level early functional deficits that may herald frank renal cellular damage. Second, other studies will evaluate the functional properties of membrane vesicles obtained from pleiotropic drug resistant cells to confirm whether there is a transport defect in these cells that accounts for drug resistance and to define the changes in the physical properties of these vesicles that may be the basis for the phenomenon.