It is our goal to investigate the biosynthesis of galactose-containing compounds in higher plants. Searching a database of expressed sequence tags, a cDNA from Arabidopsis thaliana (clone 108G20T7) with sequence similarity to UDP-glucose epimerase was identified and further analyzed. The 1356-bp-long cDNA included an open reading frame predicted to encode a 351 amino acid protein of 39 kDa. The presumed protein sequence showed a high degree of similarity to UDP-glucose epimerase sequences from bacteria, rat, and yeast. Complementation of the Saccharomyces cerevisiae gal1O mutant and expression of an active enzyme in Escherichia coli demonstrated that the cDNA encoded a functional UDP-glucose epimerase. The recombinant enzyme was purified to homogeneity. It showed a broad pH optimum of 7.0 to 9.5 and a Km of 0.11 mM. The UDP-glucose epimerase activity was not dependent on the addition of the cofactor NAD+ and was only moderately inhibited by high salt concentrations. Tissue-specific Northern analysis showed that the gene is expressed in all tissues of A. thaliana with highest expression levels in the stems and roots. Based on Southern analysis, there seems to be a single gene encoding UDP-glucose epimerase in A. thaliana. The cDNA analyzed during this study is the first known to encode a sugar-nucleotide modifying enzyme from higher plants. Its availability provides the means to investigate the role of UDP-glucose epimerase for the biosynthesis of UDP-galactose as precursor of galactolipids and cell wall polysaccharides.