We present a diagnostic question cluster (DQC) that assesses undergraduates' thinking about photosynthesis. This assessment tool is not designed to identify individual misconceptions. Rather, it is focused on students' abilities to apply basic concepts about photosynthesis by reasoning with a coordinated set of practices based on a few scientific principles: conservation of matter, conservation of energy, and the hierarchical nature of biological systems. Data on students' responses to the cluster items and uses of some of the questions in multiple-choice, multiple-true/false, and essay formats are compared. A cross-over study indicates that the multiple-true/false format shows promise as a machine-gradable format that identifies students who have a mixture of accurate and inaccurate ideas. In addition, interviews with students about their choices on three multiple-choice questions reveal the fragility of students' understanding. Collectively, the data show that many undergraduates lack both a basic understanding of the role of photosynthesis in plant metabolism and the ability to reason with scientific principles when learning new content. Implications for instruction are discussed. (Contains 7 tables and 2 figures.)