The nucleotide sequences of two highly homologous 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS; EC 220.127.116.11)-encoding genes, ACS1 and ACS3, of Arabidopsis thaliana (At) have been determined. The sequence analysis shows that ACS3 is a pseudogene representing a truncated version of ACS1. The missing region of ACS3 corresponding to the fourth exon of ACS1 has been shown by Southern analysis to be absent in the At genome. The chromosomal locations of the five members of the At ACS multigene family have been determined. The results show that each family member resides on a different chromosome. This observation suggests that the ACS3 pseudogene originated by a partial inter-chromosomal gene duplication. The ACS1 polypeptide contains all the conserved and characteristic domains found in the ACC synthase isoenzymes from various plant species, but is unable to express ACS activity in Escherichia coli and yeast. The predicted amino-acid sequence of ACS1 is missing the highly conserved tripeptide, Thr-Asn-Pro (TNP), between Ile204 and Ser205. Introduction of TNP into ACS1 restores the ACS activity, whereas its removal from the enzymatically active ACS2 results in a loss of activity. The results suggest that TNP is crucial for expression of ACS activity in E. coli.