The transcriptional activity of rodent ribosomal RNA gene (rDNA) in the primate cell was examined in the light of reported species specificity of eukaryotic ribosomal RNA synthesis. The present study showed that rat rDNA can be transcribed in HeLa nuclear extract whereas mouse rDNA was not transcribed in the heterologous extract. Rat and mouse rDNA transcription factors were interchangeable with respect to efficiency and accuracy of transcription. The initiation of rat ribosomal gene transcription by RNA polymerase I occurred at the +1 site in the heterologous extract. Initiation of transcription at the correct site also occurred in vivo following transfection of cloned rat rDNA into the primate (COS-7) cells. These data indicate that rat ribosomal RNA gene can be expressed in the primate system in vitro and in vivo. The absolute lack of species specificity in rDNA transcription has been discussed based on the present data and other reports.