Previously we have demonstrated that the U2 snRNA genes from the higher plant Arabidopsis thaliana contain two upstream elements, the USE with sequence RTCCCACATCG and a -30 'TATA' box, which are essential for transcription by RNA polymerase II, and that the conserved spacing of about four helical DNA turns between these elements is important for optimal promoter function. We have now isolated three genes encoding U6 RNA in Arabidopsis. Transcription of these genes in transfected protoplasts of Nicotiana plumbaginifolia is resistant to alpha-amanitin indicating that they are transcribed by RNA polymerase III. The upstream regions of three Arabidopsis U6 genes contain USE and -30 TATA-like elements similar to those found to be important for transcription of U2 RNA genes but the spacing between the two elements is about 10 bp closer than in the U2 genes. Using synthetic U6 genes we demonstrate that the USE and TATA elements are indispensable for their transcription, the TATA boxes of U2 and U6 genes are interchangeable, and that the intragenic A box-like sequence of U6 gene is not essential. Increasing the distance between the USE and TATA by 10 bp inactivates U6 gene transcription, demonstrating that proper positioning of the elements is also important for transcription by RNA polymerase III. The data indicate that the structure of U-snRNA gene promoters and the determinants of polymerase specificity are completely different between vertebrates and plants.