Echinocandins represent a new antifungal group with potent activity against most Candida and Aspergillus species. These lipopeptides inhibit synthesis of [beta]-1,3-glucan, a major cell wall polysaccharide. Rare acquired resistance is associated with mutations in two "hotspot" regions of Fks1 or Fks2, the probable [beta]-1,3-glucan syntheses. Intrinsic resistance of fungi such as Fusarium, Scedosporium, and Cryptococcus species represents a major limitation. Here, we explored the basis for echinocandin resistance using Saccharomyces cerevisiae as a model. Previously characterized mutations confer cross-resistance; we screened for mutations conferring differential resistance, consistent with a direct interaction of that residue with a variable echinocandin side chain. One mutant (fks1[delta] background) exhibited [greater than or equal to]16-fold differential resistance to micafungin versus caspofungin. Sequencing revealed a novel double mutation, W714/Y715N, in a previously uncharacterized region of Fks2. Equivalent (W695/Y696N) and related mutations in Fks1 confirmed the role of new "hotspot 3" in resistance. Further mutagenesis expanded hotspot 3 to residues 690-700, yielding phenotypes including differential resistance, cross-resistance, and hypersensitivity. Fks1 sequences from intrinsically resistant Scedosporium species revealed W695F-equivalent substitutions; Fks1 hybrids expressing Scedosporium prolificans hotspot 3 confirmed this substitution imparts resistance. With evidence of direct Fks1-echinocandin interaction, we turned our attention to where and how this interaction occurs. Since Fks1 is an integral membrane protein and echinocandins are cyclic peptides with lipid tails essential to their activity, protein topology is key to understanding the Fks1-echinocandin interaction. To address this, we used a Suc2/His4C reporter fused to a series of C-terminal truncations to experimentally define Fks1 topology in S. cerevisiae. Of the 15-18 transmembrane helices predicted in silico, 13 were experimentally confirmed. The N-terminus (residues 1-445) is cytosolic and the C-terminus (residues 1823-1876) external; both are important for Fks1 activity. The cytosolic central domain (residues 715-1294) includes newly recognized homology to glycosyltransferases; residues potentially involved in catalysis and UDP-glucose binding are essential. Importantly, all three hotspots are external, with hotspots 1 and 2 adjacent to and hotspot 3 largely embedded within the outer leaflet of the membrane. This topology suggests a model where echinocandins interact through their lipid tails with hotspot 3 and through their cyclic peptides with hotspots 1 and 2.