A large-scale paper-and-pencil assessment of the attainment targets of environmental studies with a focus on the subject area nature was held in primary education in Flanders (Belgium). The tests on different subfields of nature, i.e. the human body, healthcare, organisms, ecosystems, environmental care and non-living nature, were administered to a representative sample of 4556 pupils of the sixth grade from 145 different schools. The percentage of students mastering the attainment targets differed clearly across the tested subfields. Moreover, gender differences were found. Multilevel analyses revealed that 13% of the total variance of the performance on an overall scale could be attributed to differences between schools. For 82%, these school differences were accounted for by factors mainly referring to the language spoken at home and to the socio-economic background of the pupils. Differences among pupils within schools were explained only to a small extent. (Contains 1 figure and 5 tables.)