The successful use of mixed heterologous in vitro transcription systems has suggested that the species specificity of RNA polymerase III transcription is low. To see if this extends to lower eukaryotic class III transcription factors, we compared the interactions of the two yeast assembly factors, TFIIIA and TFIIIC/tau factor, with a homologous yeast 5S rRNA gene and a heterologous Xenopus laevis somatic 5S rRNA gene. Transcription assays showed that the Xenopus gene was transcriptionally inactive in a crude cell-free yeast extract that actively transcribes the homologous gene. However, the Xenopus gene was still able to compete for limiting transcription factors. Electrophoretic DNA binding assays revealed that while TFIIIA bound avidly to the yeast gene (generating the 'A-complex'), it had no affinity for the Xenopus 5S rRNA gene. Nevertheless, a complex of both TFIIIA and TFIIIC/tau factor (the 'AC-complex') was formed on the two genes with similar affinity, although only the complex assembled on the homologous gene was able to activate transcription. Thus enough sequence information is present on the heterologous gene to direct transcription factor assembly, but not to activate transcription. Like its counterpart in Xenopus, the yeast TFIIIA appears to be a zinc binding protein that is inactivated by EDTA and 1,10-phenanthroline, and reactivated in the presence of zinc ions. Bound to the 5S rRNA gene, TFIIIA is however significantly more resistant to inactivation by chelators than in its free state. The AC-complex differs from the A-complex by being less affected by chelators, and by being more sensitive to the dissociating effect of single-stranded DNA.