The use of assessment methods is a widely discussed topic in education. Exams that are commonly used, however, may not necessarily assess student understanding at a high cognitive level. By implementing alternative assessment methods, instructors can influence student learning. Portfolio-based assessment (PBA) is a purposeful collection of student work that represents achievement, progress, growth, and reflection. In this study, we examined student PBA that has been implemented in an upper-level biology course. The two sections of the portfolios analyzed were students' written reflections and questions from the inquiry section. Students' knowledge of biological concepts was also assessed on an initial and final assessment. Although initially poor, students' knowledge increased substantially on the final assessment. The majority of student reflections were found to be naive or novice in quality, and no significant difference was found in quality nor between topics covered. Variation, however, existed between topics on which students chose to reflect. In the inquiry section, students posed an almost equal number of higher and lower cognitive-level questions. This study is one of the first examining PBA in an upper-level college science course. Implementation of PBA may be a method to improve student understanding of biological concepts. (Contains 7 tables and 2 figures.)