The predicted mature portion of a putative 3-hydroxyacyl-ACP dehydratase (DH) from Arabidopsis was linked to an N-terminal poly-histidine-tag and the fusion protein expressed in Escherichia coli. Soluble dehydratase was present on induction at 25 degrees C and pure dehydratase eluted from a nickel-affinity column in 0.2-0.5M imidazole. High concentrations of imidazole were necessary to retain enzyme solubility. The dehydratase reaction is reversible and 3-hydroxybutyryl- and 2-butenoyl-ACP substrates were prepared from E. coli apo-ACP. Analysis of these suggested contamination of apo-ACP with dehydratase and an additional reverse-phase chromatographic step was required during acyl carrier protein (ACP) preparation. Activity of purified dehydratase was demonstrated by mass spectrometry using 2-butenoyl-ACP, providing the first functional experimental evidence for plant DH gene sequences.