SummaryMany plants increase in freezing tolerance in response to low temperature, a process known as cold acclimation. In Arabidopsis, cold acclimation involves action of the CBF cold response pathway. Key components of the pathway include rapid cold‐induced expression of three homologous genes encoding transcriptional activators, CBF1, 2 and 3 (also known as DREB1b, c and a, respectively), followed by expression of CBF‐targeted genes, the CBF regulon, that increase freezing tolerance. Unlike Arabidopsis, tomato cannot cold acclimate raising the question of whether it has a functional CBF cold response pathway. Here we show that tomato, like Arabidopsis, encodes three CBF homologs, LeCBF1–3 (Lycopersicon esculentum CBF1–3), that are present in tandem array in the genome. Only the tomato LeCBF1 gene, however, was found to be cold‐inducible. As is the case for Arabidopsis CBF1–3, transcripts for LeCBF1–3 did accumulate in response to mechanical agitation, but not in response to drought, ABA or high salinity. Constitutive overexpression of LeCBF1 in transgenic Arabidopsis plants induced expression of CBF‐targeted genes and increased freezing tolerance indicating that LeCBF1 encodes a functional homolog of the Arabidopsis CBF1–3 proteins. However, constitutive overexpression of either LeCBF1 or AtCBF3 in transgenic tomato plants did not increase freezing tolerance. Gene expression studies, including the use of a cDNA microarray representing approximately 8000 tomato genes, identified only four genes that were induced 2.5‐fold or more in the LeCBF1 or AtCBF3 overexpressing plants, three of which were putative members of the tomato CBF regulon as they were also upregulated in response to low temperature. Additional experiments indicated that of eight tomato genes that were likely orthologs of Arabidopsis CBF regulon genes, none were responsive to CBF overexpression in tomato. From these results, we conclude that tomato has a complete CBF cold response pathway, but that the tomato CBF regulon differs from that of Arabidopsis and appears to be considerably smaller and less diverse in function.