In the GENE experiment performed during an 11-day Soyuz Mission to the International Space Station (ISS), we intended to determine if microgravity aff ects Drosophila metamorphosis processes. Control experiments were performed including a 1g ground control parallel to the ISS flight samples and a Random Position Machine microgravity simulated control. A preliminary analysis of the results indicates that five hundred to one thousand genes change their expres- sion profiles depending on the cut-off levels selected. Especially affected among them are the mitochondrial ones (an example with the respiratory chain is pre- sented). We show here that there is a synergic effect of the constraints introduced to meet the requirements of the space experiment (mainly, a cold step and the use of hermetically closed Type-I containers). The cold transport step to the launch site was introduced to slow down the pupal development. The hermetically closed Type I containers were required to ensure the containment of the fixative (acetone) in the experiment. As shown here, the oxygen concentration inside the container was not optimal but fully compatible with pu- pal development. It is highly likely that such combined environmental effects will become a common finding in these types of studies as they become more complicated and extensive. They could open the way to understand how the gene expression patterns and the actual phe- notypes can adjust to the environment. These findings indicate the importance of a vigorous ground based program in support of real microgravity experiments. Only then we can utilize the ISS in order to understand the consequences of the modified environment in outer space on living organisms.