The Arabidopsis thaliana Srp54 gene family was determined to consist of three genes, all of which were cloned and sequenced. In addition, cDNAs corresponding to two of the genes were obtained. To our knowledge this is the first description of multiple Srp54 genes within an organism. In contrast to the situation in mammals, where there are only three amino acid differences between the mouse and canine sequences, there was significant amino acid sequence diversity among the genes, particularly in the methionine-rich region of the protein, which is the region responsible for binding to the 7S RNA of the signal recognition particle and to the signal sequence of newly synthesized proteins. The amino acid sequences of the GTP-binding domains of the three clones were 86% identical, whereas the methionine-rich domains were only 65% identical. RNA gel blots of various tissues and developmental stages hybridized with gene-specific probes revealed that all three genes were expressed in all the tissues investigated. There were, however, quantitative differences in expression levels.