A cDNA clone encoding a histidyl-tRNA synthetase (HisRS) was characterized from Arabidopsis thaliana. The deduced amino acid sequence (AtHRS1) is surprisingly more similar to HisRSs from archaebacteria than those from eukaryotes and prokaryotes. AtHRS1 has an N-terminal extension with features characteristic of mitochondrial and chloroplast transit peptides. Transient expression assays in tobacco protoplasts clearly demonstrated efficient targeting of a fusion peptide consisting of the first 71 amino acids of AtHRS1 joined to jellyfish green fluorescent protein (GFP) to both mitochondria and chloroplasts. These observations suggest that the AtHisRS1 cDNA encodes both mitochondrial and chloroplast histidyl-tRNA synthetases.