Leafy spurge (Euphorbia esula) is a perennial weed which is capable of acclimating to sub-freezing temperatures. We have used the differential display technique to identify and clone a cDNA for a cold-regulated gene (cor20) which hybridizes to mRNAs that accumulate specifically during the cold acclamation process. The cor20 cDNA was used to isolate two different genomic clones. Both clones were similar but not identical to each other and the cDNA. Sequence analysis of the genomic clones indicated that they share considerable homology to a group of glycine-rich RNA-binding protein genes. Comparison of the promoter region from the three clones (Ccr1 from Arabidopsis, BnGRP1O from Brassica napus, and GRRBP2 from Euphorbia esula) have identified at least two conserved motifs. CAGC is most likely involved in cold regulation and AACCCYAGTTA, is conserved but has no known function. RNAs which hybridize to cor20 reach maximal expression in less than 2 days after exposure of the plant to temperatures of 5 °C, and remains at high levels in the plant for at least 30 days so long as the plant is left in the cold. These RNAs drop to control levels within 24 h when the plant is returned to normal growing temperatures. Transcripts which hybridize to cor20 do not accumulate under conditions of drought or heat stress. These transcripts are induced in response to low temperatures in roots, stems and leaves, but are expressed constitutively in tissue culture at control temperatures.