Reconstitution experiments showed that the two Escherichia coli 5S RNA binding proteins L18 and L25 form a specific complex with yeast 5.8S RNA and not with yeast 5S RNA. The yeast 5.8S RNA-E. coli protein complex was found to exhibit ATPase and GTPase activities that had previously been observed for the E. coli 5S RNA-protein complex. The tetranucleotide UpUpCpG, which is an analog of the tRNA fragment TpsipCpG, interacted strongly with 5S RNA-protein complexes from E. coli and Bacillus stearothermophilus and weakly with yeast 5.8S RNA. UpUpCpG did not bind to E. coli, B. stearothermophilus, or yeast 5S RNA or to the yeast 5.8S RNA-E. coli protein complex. It is suggested that 5.8S RNA evolved from prokaryotic 5S RNA and that the latter two RNAs are related and have similar functions in protein synthesis.