This study examines how the use of self- and peer-assessment within a girls-only biology class can support students' motivation. The study took place over 22 weeks in a rural comprehensive school, and the participants were girls between 15 and 16 years of age. Data included questionnaires, semi-structured interviews, notes from lesson observations and video-transcripts of peer-assessment episodes. Data analysis suggests that girls' motivation may be supported, both by being taught in a single-sex group and by employing assessment for learning techniques. However, such benefits were not ubiquitous and may have been negated by examination anxiety, inadequate time to adapt to assessment for learning strategies, limited negative effects of single-sex teaching on learning, the way in which the single-sex class was created and some girls' resilient conceptions of science being a masculine subject.