Arabidopsis thaliana shows hybrid vigor (heterosis) in progeny of crosses between Columbia-0 and C24 accessions. Hybrid vigor was evident as early as mature seeds and in seedlings 3 d after sowing (DAS). At 3 DAS, genes encoding chloroplast-located proteins were significantly overrepresented (187) among the 724 genes that have greater than midparent values of expression in the hybrid. Many of these genes are involved in chlorophyll biosynthesis and photosynthesis. The rate of photosynthesis was constant per unit leaf area in parents and hybrids. Larger cell sizes in the hybrids were associated with more chloroplasts per cell, more total chlorophyll, and more photosynthesis. The increased transcription of the chloroplast-targeted genes was restricted to the 3-7 DAS period. At 10 DAS, only 118 genes had expression levels different from the expected midparent value in the hybrid, and only 12 of these genes were differentially expressed at 3 DAS. The early increase in activity of genes involved in photosynthesis and the associated phenomena of increases in cell size and number through development, leading to larger leaf areas of all leaves in the hybrid, suggest a central role for increased photosynthesis in the production of the heterotic biomass. In support of this correlation, we found that an inhibitor of photosynthesis eliminated heterosis and that higher light intensities enhanced both photosynthesis and heterosis. In hybrids with low-level heterosis (Landsberg erecta x Columbia-0), chloroplast-targeted genes were not up-regulated and leaf areas were only marginally increased.