Anabaena sp. PCC 7120 is a filamentous cyanobacterium that bears a cluster of 26 tRNA genes and pseudogenes in the delta plasmid. The sequences of these tRNAs suggest that they have been acquired by horizontal gene transfer from another organism. The cluster is transcribed as a single transcript that is quickly processed to individual tRNAs. RNase P and RNase Z, in vitro, are able to process precursors containing some of these tRNAs. Deletion of the cluster causes no obvious phenotype or effect on growth under diverse culture conditions, indicating that the tRNAs encoded in the cluster are not required for growth under laboratory conditions, although they are aminoacylated in vivo. We have studied a possible tRNASer [tRNASerGCU(2)] present in the cluster with a sequence that deviates from consensus. This tRNA is processed in vitro by RNase P at the expected position. In addition, this tRNASerGCU is specifically aminoacylated with serine by an Anabaena sp. PCC 7120 crude extract. These data indicate that tRNASerGCU(2) is fully functional, despite its unusual structure. Similar clusters are found in other three cyanobacteria whose genomes have been sequenced.