A regulatory element upstream of the human myoglobin gene functions as a muscle-specific enhancer (MSE) in conjunction with core promoter elements of the myoglobin gene, but not in combination with the simian virus 40 (SV40) early promoter. These two promoters differ in the sequences of their 'TATA boxes': for the myoglobin gene, the sequence is TATAAAA, whereas for SV40, the sequence is TATTTAT. We have now tested the hypothesis that this sequence difference is responsible for the differential response of the promoters to the MSE. We found that when the TATA box sequence of the myoglobin promoter was changed to that of the SV40 promoter, responsiveness to the MSE was abolished; conversely, when the SV40 TATA box sequence was changed to that of the myoglobin promoter, the promoter became responsive to the MSE. We conclude that mammalian TATA-box elements are functionally heterogeneous, and suggest that this heterogeneity reflects differential interactions with distinctive TATA box-binding factors, only some of which can act cooperatively with MSE-binding proteins to generate an active transcriptional complex.