Technical Abstract: Patulin, a secondary metabolite produced by several fungal species, is a potential contaminant of fruit and vegetable products. To better understand the structure and electronic properties of this mycotoxin and its biosynthetic precursors, a density functional theory (DFT) study was performed on conformations of patulin, ascladiol, and neopatulin. Geometry optimization and transition state calculations were carried out using the three parameter B3LYP functional at the 6-311++G** level of theory. Both aqueous solvation studies using a continuum solvation model and in vacuo calculations resulted in several stable conformations of patulin within a range two kcal/mol. One conformation was preferred by ~one kcal/mol over the other geometry optimized conformations considered. Similar results were found for neopatulin. Ascladiol had several stable conformations within two kcal/mol that possessed favorable intramolecular hydrogen bond interactions. High level density functional calculations were applied to investigate the conformational preferences of important molecules involved in patulin synthesis, and these results serve as a basis for modeling studies and interpretation of experimental results.