Two leucine tRNAs from the cyanophyte Anacystis nidulans have been isolated, and their complete nucleotide sequences have been determined by combining data from oligonucleotide fingerprints and sequencing gels. The two sequences are 87 nucleotides long, have the anticodons CAA and CAG, and differ from each other at a total of 28 positions. They have been compared to other known tRNA Leu sequences and incorporated into a phylogenetic tree comprising prokaryotic and chloroplastic tRNA Leu sequences. Mutations inferred from the tree show that some parts of the tRNA molecule are highly variable (the extra arm and the acceptor stem) while others are much more conserved (the D and T arms). The topology of the tree supports the idea that blue-green algae and chloroplasts share a common prokaryotic ancestor and show a basic divergence between XAA and XAG anticodon-containing tRNAs, suggesting that these two subfamilies result from an ancient gene duplication. Finally, comparison of this phylogenetic tree with those of other multi-isoacceptor tRNA families shows no common scheme, which may be due to independent refinement of codon-reading patterns in different tRNA families.