GEK1, an Arabidopsis thaliana gene product, was recently identified through its involvement in ethanol tolerance. Later, this protein was shown to display 26% strict identity with archaeal D-Tyr-tRNATyr deacylases. To determine whether it actually possessed deacylase activity, the product of the GEK1 open reading frame was expressed in Escherichia coli from a multi-copy plasmid. Purified GEK1 protein contains two zinc ions and proves to be a broad-specific, markedly active D-aminoacyl-tRNA deacylase in vitro. Moreover, GEK1 expression is capable of functionally compensating in E. coli for the absence of endogeneous D-Tyr- tRNATyr deacylase. Possible connections between exposure of plants to ethanol/acetaldehyde and misaminoacylation of tRNA by D-amino acids are considered.