The transcriptome of seedlings was analyzed from experiments performed on the International Space Station to study the interacting effects of light and gravity on plant tropisms (project named TROPI-2; Kiss et al. 2012). Seeds of Arabidopsis were germinated in space, and seedlings were then grown in the European Modular Cultivation System for 4 days at ~1g followed by exposure to a range of gravitational accelerations (from microgravity to 1g) and two light treatments (blue light with or without a 1 h pretreatment with red). At the end of the experiments, the cassettes containing the seedlings were frozen in the minus eighty laboratory freezer and returned to Earth on space shuttle mission STS-131. The RNA was extracted from whole seedlings and used for the transcriptome analyses. A comparison of 1g spaceflight samples with 1g ground controls identified 230 genes that were differentially regulated at least twofold, emphasizing the need for "in situ" tissue fixation on a 1g centrifuge as an important control for spaceflight experiments. A further comparison of all spaceflight samples with ground controls identified approximately 280 genes that were differentially regulated at least twofold. Of these genes, several were involved in regulating cell polarity (i.e., auxin, calcium, lipid metabolism), cell-wall development, oxygen status, and cell defense or stress. However, when the transcriptome of the all g-treated spaceflight samples was compared with microgravity samples, only ~130 genes were identified as being differently regulated (P ≤ 0.01). Of this subset, only 27 genes were at least twofold differently regulated between microgravity and 1g space samples and included putative/pseudo/undefined genes (14), transposable elements (5), an expansin (ATEXP24; At1g21240), a cell-wall kinase (WAK3; At1g21240), a laccase-like flavonoid oxidase (TT10; At5g48100), among others.