Despite the significant contributions of utilizing heterosis to crop productivity worldwide, the biological mechanisms of heterosis remained largely uncharacterized. In this study, we analyzed gene expression profiles of an elite rice hybrid and the parents at three stages of young panicle development, using a cDNA microarray consisting of 9198 expressed sequence tags (ESTs), with the objective to reveal patterns of gene expression that may be associated with heterosis in yield. A total of 8422 sequences showed hybridization signals in all three genotypes in at least one stage and 5771 sequences produced detectable signals in all slides. Significant differences in expression level were detected for 438 sequences among the three genotypes in at least one of the three stages, as determined by ANOVA validated with 100 permutations at P < 0.05. Significant mid-parent heterosis was detected for 141 sequences, which demonstrated the following features: a much larger number of sequences showed negative heterosis than ones showing positive heterosis; genes functioning in DNA replication and repair tended to show positive heterosis; genes functioning in carbohydrate metabolism, lipid metabolism, energy metabolism, translation, protein degradation, and cellular information processing showed negative heterosis; both positive and negative heterosis were observed for genes in amino acid metabolism, transcription, signal transduction, plant defense and transportation. The results are indicative of the biochemical and physiological activities taking place in the hybrid relative to the parents. Identification of genes showing expression polymorphisms among different genotypes and heterotic expression in the hybrid may provide new avenues for exploring the biological mechanisms underlying heterosis.