Within the framework of cognitive learning theories, instructional design manipulations have primarily been investigated under tightly controlled laboratory conditions. We carried out two experiments, where the first experiment was conducted in a restricted system-paced setting and is therefore in line with the majority of empirical studies in the learning sciences. However, the second experiment was done in an ecologically more valid classroom setting, with students working at their own pace with the instructional material embedded in a professional hypermedia learning environment. Both dealt with the same topic in the domain of biological education, namely the structure and functioning of the enzyme ATP-Synthase. In both experiments, the educational value of three- versus two-dimensional animations as well as of visual cues was investigated in a 2 x 2 factorial design. Students' understanding was facilitated by the presence of a 3D-representation format under tightly controlled conditions only. Regarding the ecologically more valid classroom setting, the 2D format tended to foster understanding more efficiently than the 3D format. The implementation of visual cues enhanced the amount students remembered in both experiments. Our results indicate that the results of tightly controlled laboratory conditions may not be easily generalized to naturalistic classroom settings.