Prior research states that the use of living animals in the classroom leads to a higher knowledge but those previous studies have methodological and statistical problems. We applied a meta-analysis and developed a treatment-control study in a middle school classroom. The treatments (film vs. living animal) differed only by the presence of the living animal. Both treatments were based on the self-determination theory. More than 400 pupils filled in pre-test, post-test and two follow-up-tests (with a delay of 6-8 weeks and 7-8 months). After each lesson, pupils rated the lesson on a short intrinsic motivation scale. In the meta-analysis, we found that the living animal treatments significantly scored better than a control group, but not when comparing living animals with alternative treatments. In the treatment-control study, both treatments led to a significant increase in knowledge but there were no differences between film and living animal treatment. Pre-test and previous grading had a significant influence on post- and both follow-up tests. In the mouse lesson, pupils of the living animal group showed higher values in interest and competence and lower values in pressure. Interest and competence correlated positively with achievement, while pressure correlated negatively.