The molecular characterization of two isoforms of 3-deoxy-d-manno-oct-2-ulosonate (KDO) -8-phosphate synthase (AtkdsA1 and AtkdsA2) from Arabidopsis is reported here. First, by isolating a full-length cDNA for AtkdsA1, it was confirmed that the deduced primary structures of AtkdsA1 and AtkdsA2 proteins were 93% identical. Functional expression and purification studies demonstrated the efficient catalytic activity of the AtkdsA1 enzyme to produce KDO-8-phosphate from phosphoenolpyruvate and d-arabinose-5-phosphate. RT-PCR and RNA-gel blot analysis revealed different expression profiles for both genes; the AtkdsA1 gene was predominantly expressed in the shoots, while the AtkdsA2 transcript accumulated to a higher level in the roots, implicating differential roles of these isoforms in planta.