Xyloglucan endotransglycosylases (XETs) modify a major component of the plant cell wall and therefore may play critical roles in generating tissue properties and influencing morphogenesis. An XET-related gene family exists in Arabidopsis thaliana, the members of which show differential regulation of expression. TCH4 expression is rapidly regulated by mechanical stimuli, temperature shifts, light, and hormones. As a first step in determining whether Arabidopsis XET-related proteins have distinct properties, we produced recombinant TCH4 protein in bacteria and determined its enzymatic characteristics. TCH4 specifically transglycosylates only xyloglucan. The enzyme prefers to transfer a portion of a donor polymer onto another xyloglucan polymer (acceptor); TCH4 will also utilize xyloglucan-derived oligosaccharides as accepters but discriminates between differentially fucosylated oligosaccharides. TCH4 is most active at pH 6.0 to 6.5 and is surprisingly cold-tolerant with an optimum of 12 to 18 degrees C. TCH4 activity is enhanced by urea and bovine serum albumin, but not cations, reducing agents, or carboxymethylcellulose. These studies indicate that TCH4 is specific for xyloglucan, but that the molecular mass and the fucosyl content of the substrates influence enzymatic reaction rates. TCH4 is unlikely to play a role in acid-induced wall loosening but may function in cold acclimation or cold-tolerant growth.