The Xenopus laevis selenocysteine tRNA[Ser]Sec gene utilizes the TATA box binding protein (TBP) for its transcription in a manner more like TATA-dependent class II genes than TATA-less class III tRNA genes, even though this gene is transcribed by RNA polymerase III (Pol III). Addition of TBP increased in vitro transcription of the tRNA[Ser]Sec gene and a RNA polymerase II-(Pol II-) dependent template, while it decreased TATA-independent tRNA(Met) gene transcription, in a dose-dependent manner. Addition of wild-type TBP, truncated TBP containing the highly conserved COOH-terminal domain or a mutant TBP defective in TATA-independent Pol III transcription to TBP-depleted extracts restored tRNA[Ser]Sec transcription, while addition of a mutant TBP defective in Pol II transcription did not. These studies provide evidence that common surfaces of TBP may be used in transcription from TATA-dependent promoters of the tRNA[Ser]Sec gene and class II genes. Further, we show that distinct chromatographic fractions of TBP complexes are required for tRNA[Ser]Sec gene transcription and TATA-less class III gene transcription.