We have established the minimal sequence and factor requirements for both constitutive and viral-induced transcription from an atypical, TATATAA box-containing human housekeeping promoter. Utilizing a transient cotransfection protocol, we have found that efficient transactivation of triosephosphate isomerase (TPI) gene transcription by the immediate early proteins of adenovirus and pseudorabies virus is dependent upon the same assembly of sequence elements that collectively confer minimal TPI promoter function in the absence of viral protein. These elements span TPI promoter positions -65 and -6 (where +1 is the transcription initiation site) and include not only a TFIID-responsive TATATAA box (-27 to -21) but a single GC box (-53 to -48) that binds Spl, and a novel cap proximal element (-18 to -6) that binds a 110-kDa nuclear factor that is present in HeLa cells. We demonstrate that these elements function in an interdependent fashion; deleting either GC box 1 or the cap proximal element completely or nearly abolished both basal transcription and viral transactivation. Therefore, these elements and their cognate factors represent the basal transcription initiation complex through which the immediate early protein of adenovirus or pseudorabies virus mediates the stimulation of TPI gene transcription. We discuss the implications of these data for both constitutive and viral-induced transcription.