The TATA box, located upstream at about nt -30, and the proximal sequence element, located at about nt -60, are both essential and sufficient for basal level transcription of the Xenopus laevis (Xl) selenocysteine (Sec) tRNA[Ser]Sec gene as demonstrated by its microinjection into Xl oocytes. Point mutations within either of these regions abolish transcription, while deletion of the internal boxA element or insertion of 13 nt within the internal boxB element does not impair transcription. The latter mutations (within the internal regions) affect processing of the 3'-trailer sequence. Replacement of the tRNA[Ser]Sec coding sequence with an Escherichia coli M1 RNA gene resulted in expression of the E. coli gene governed by the upstream tRNA[Ser]Sec promoter elements. These studies demonstrate unequivocally that the upstream promoter elements are sufficient for the basal level of tRNA[Ser]Sec gene transcription. Primer extension studies with spacer mutants show that the internal elements do not play a role in selecting the transcription start point (tsp), but that selection of the tsp is determined by the region upstream from the gene. Further, studies with spacer mutants show that the distance between the TATA box and the tsp is quite likely the critical factor in selecting the position of tsp.