We have investigated the contribution of specific TATA-binding protein (TBP)-TATA interactions to the promoter activity of a constitutively expressed silkworm tRNA_C^Ala gene and have also asked whether the lack of similar interactions accounts for the low promoter activity of a silk gland-specific tRNA_SG^Ala gene. We compared TBP binding, TFIIIB-promoter complex stability (measured by heparin resistance), and in vitro transcriptional activity in a series of mutant tRNA_C^Ala promoters and found that specific TBP-TATA contacts are important for TFIIIB-promoter interaction and for transcriptional activity. Although the wild-type tRNA_C^Ala promoter contains two functional TBP binding sequences that overlap, the tRNA_SG^Ala promoter lacks any TBP binding site in the corresponding region. This feature appears to account for the inefficiency of the tRNA_SG^Ala promoter since provision of either of the wild-type TATA sequences derived from the tRNA_C^Ala promoter confers robust transcriptional activity. Transcriptional impairment of the wild-type tRNA_SG^Ala gene is not due to reduced incorporation of TBP into transcription complexes since both the tRNA_C^Ala and tRNA_SG^Ala promoters form transcription complexes that contain the same amount of TBP. Thus, the deleterious consequences of the lack of appropriate TBP-TATA contacts in the tRNA_SG^Ala promoter must come from failure to incorporate some other essential transcription factor(s) or to stabilize the complete complex in an active conformation.