Background: Establishing or preserving single-sex schooling has been widely discussed as a way of bringing more girls into the natural sciences. Aims: We test the assumption that the beneficial effects of single-sex education on girls' self-concept of ability in masculine subjects such as physics are due to the lower accessibility of gender-related self-knowledge in single-sex classes. Sample: N401 eighth-graders (mean age 14.0 years) from coeducational comprehensive schools. Methods: Random assignment of students to single-sex vs. coeducational physics classes throughout the eighth grade. At the end of the year, students' physics-related self-concept of ability was measured using a questionnaire. In a subsample of N134 students, the accessibility of gender-related self-knowledge during physics classes was assessed by measuring latencies and endorsement of sex-typed trait adjectives. Results: Girls from single-sex physics classes reported a better physics-related self-concept of ability than girls from coeducational classes, while boys' self-concept of ability did not vary according to class composition. For both boys and girls, gender-related self-knowledge was less accessible in single-sex classes than in mixed-sex classes. To the extent that girls' feminine self-knowledge was relatively less accessible than their masculine self-knowledge, their physics-related self-concept of ability improved at the end of the school year. Conclusions: By revealing the importance of the differential accessibility of gender-related self-knowledge in single- and mixed-sex settings, our study clarifies why single-sex schooling helps adolescents to gain a better self-concept of ability in school subjects that are considered inappropriate for their own sex.