Molecular genetics has identified dozens of genes that regulate flower development in Arabidopsis. However, the complexity of flower development suggests that many other genes are yet to be uncovered. To identify floral genes that are expressed at low levels in the flower, we have sequenced 1587 cDNA fragments from a subtractive floral cDNA library. A total of 1222 unique genes represented by these ESTs are distributed on all five chromosomes with similar frequencies as all predicted genes in the genome. Among these, 17 genes were shown to be expressed anywhere for the first time because they were not found in previous EST and full-length cDNA datasets. Furthermore, 724 of the genes revealed by this library were not definitively shown to be expressed in the flower by previous floral EST datasets. In addition, 49 transcriptional regulators, 31 protein kinases, 12 zinc-finger proteins and other signaling proteins were found to be present in floral buds. Moreover, the EST sequences likely extended the transcribed regions of 26 previously annotated genes, and may have uncovered several previously unrecognized genes. To obtain additional clues about possible gene function, we hybridized cDNA microarray with probes derived from wild-type Arabidopsis rosette leaves and floral buds. We estimated that over 50% of genes were expressed at levels lower than 1/30 of the highest detectable signal intensity, indicating that many floral genes are expressed at low levels. Furthermore, 97 genes were found to be expressed at a higher level in the flower than the leaf by the Significance Analysis of Microarray (SAM) method with a 1.0% false discovery rate (FDR). Further RT-PCR analyses of selected genes support the microarray results. We suggest that the genes encoding putative regulatory proteins and at least some proteins with currently unknown functions might play important roles during flowering.