Students commencing studies in biochemistry must transfer and build on concepts they learned in chemistry and biology classes. It is well established, however, that students have difficulties in transferring critical concepts from general chemistry courses; one key concept is “energy.” Most previous work on students' conception of energy has focused on their understanding of energy in the context of physics (including the idea of “work”) and/or their understanding of energy in classical physical and inorganic chemistry contexts (particularly Gibbs Free Energy changes, the second law of thermodynamics, and equilibrium under standard conditions within a closed system). For biochemistry, students must go beyond those basic thermodynamics concepts of work, standard energy changes, and closed systems, and instead they must consider what energy flow, use, and transformation mean in living, open, and dynamic systems. In this study we explored students' concepts about free energy and flow in biological chemical reactions and metabolic pathways by surveys and in-depth interviews. We worked with students in general chemistry classes and biochemistry courses in both an Australian and a US tertiary institution. We address three primary questions (i) What are the most common alternative conceptions held by students when they explain energy-related phenomena in biochemistry?, (ii) What information do students transfer from introductory chemistry and biology when they are asked to consider energy in a biological reaction or reaction pathway?, and (iii) How do students at varying levels of competence articulate their understandings of energy in pathways and biological reactions? The answers to these questions are used to build a preliminary learning progression for understanding “energy” in biochemistry. We also propose crucial elements of content knowledge that instructors could apply to help students better grasp this threshold concept in biochemistry.