To gain a better understanding of changes in gene expression associated with cold acclimation in the woody perennial blueberry (Vaccinium corymbosum L.) and ultimately use this information to develop more freeze-tolerant cultivars, a genomics approach based on the analysis of expressed sequence tags (ESTs) and microarrays was undertaken. Initially, two standard cDNA libraries, constructed using RNA from cold-acclimated (CA) and nonacclimated (NA) floral buds of the blueberry cultivar Bluecrop, were used for the generation of ≈2400 ESTs, half from each library. Putative functions were assigned to cDNAs based on homology to other genes/ESTs from GenBank. From contig analyses, 796 and 865 unique transcripts were identified from the CA and NA libraries, respectively. The most highly abundant cDNAs, that were picked many more times from one library than from the other, were identified as representing potentially differentially expressed transcripts. A cDNA microarray was constructed and used to study gene expression under cold-acclimating conditions in the field and cold room. Results indicated that the abundance of transcripts of numerous blueberry genes change during cold acclimation, including genes not found previously to be cold-responsive in Arabidopsis, and, interestingly, more transcripts were found to be upregulated under cold room conditions than under field conditions. Finally, forward and reverse subtracted cDNA libraries were prepared from ‘Bluecrop’ RNA to enrich for transcripts that are expressed at higher levels in floral buds at 400 h and at 0 h of low-temperature exposure, respectively. Many genes encoding putative transcription factors and other proteins related to signal transduction were identified from both libraries.