Since its discovery, it was appreciated that the antifolate pemetrexed had multiple targets within folate metabolism. This laboratory was instrumental in showing that pemetrexed elicited its primary action as a thymidylate synthase inhibitor. Unusual for an antifolate, pemetrexed showed significant clinical activity against malignant pleural mesothelioma and non-small cell lung cancer. Accordingly, the FDA recently issued first-line approvals for pemetrexed in these diseases, leading us to question whether the effects of pemetrexed on other folate-dependent targets could explain this atypical clinical activity of the drug. Studies in this dissertation showed that in addition to thymidylate synthase inhibition, pemetrexed was also an inhibitor of aminoimidazolecarboxamide ribonucleotide formyltransferase (AICART), the second folate-dependent enzyme of de novo purine synthesis. Consequent of AICART inhibition, pemetrexed caused robust activation of a key energy-sensing regulatory enzyme of the PI3K-AKT signal transduction pathway, AMP-activated protein kinase (AMPK). AMPK activation resulted from accumulation of the AMP-mimetic, ZMP, behind the AICART block. Constituents of the PI3K-AKT cascade are frequently deregulated in human carcinomas, uncoupling nutrient supply from proliferative capacity. Therefore, interventions that reinstate control over aberrant signaling along this axis, such as AMPK activation, are of significant cancer therapeutic interest. The cellular consequences of AMPK activation in response to pemetrexed were assessed. In particular, effects on the downstream target of PI3K-AKT signaling, the mammalian target of rapamycin complex 1 (mTORC1), were studied. Unlike targeted mTORC1 inhibitors, such as rapamycin and its analogs, pemetrexed-mediated activation of AMPK also signaled to mTOR-independent controlling elements of protein and lipid synthesis, highlighting additional benefits of AMPK activating agents that extend beyond effects on mTOR signaling. We therefore propose that the unusual activity of pemetrexed in mesothelioma and non-small cell lung cancer is due in part to effects on signaling processes downstream of AMPK activation. These findings present a novel approach to AMPK activation secondary to an AICART block, define pemetrexed as a molecularly targeted agent, and ultimately extend the utility of antifolates beyond their traditional function.