Biochemistry is a visual discipline that requires students to develop an understanding of numerous representations. However, there is very little known about what students actually understand about the representations that are used to communicate ideas in biochemistry. This study investigated biochemistry students' understanding of multiple representations of enzyme-substrate interactions through both student interviews (N?=?25) and responses by a national sample (N?=?707) to the Enzyme-Substrate Interactions Concept Inventory. This manuscript reports the findings regarding one category of misconceptions measured by the concept inventory, namely, students' understandings of shape and charge in the context of enzyme-substrate interactions. Students interpret molecular representations depicting such interactions by determining the complementarity between enzyme and substrate by focusing upon charge and hydrogen bonding, but with a disregard for stereochemistry.