Cytomegalovirus infection is a major cause of morbidity in immunocompromised patients. The major immediate early promoter/enhancer (MIEP) of the human cytomegalovirus controls the expression of the immediate early genes 1 and 2 which play a central role both in primary and reactivated human cytomegalovirus (HCMV)-infections. Our previous studies have shown that co-infection of A549 cells with human cytomegalovirus and human polyomavirus BK resulted in enhanced expression of the immediate early genes 1 and 2 and that the early gene products of BK virus trans-activated the MIEP. However, neither the MIEP sequences required for mediating this trans-activation, nor the contribution of the individual BK virus early gene products were examined. The MIEP contains multiple binding sites for the transcription factors CREB, AP1, Sp1 and NFkappaB, which may mediate polyomavirus large T- or small t-antigens-induced promoter activation. Transient transfection studies in A549 cells demonstrated that SV40 large T-antigen, but not small t-antigen, trans-activated MIEP activity approximately 9-fold. Mutations in individual binding motifs in the context of the complete MIEP did not impair traits-activation by large T-antigen. The level of induction of a truncated MIEP consisting of a single set of CRE/AP1, NFkappaB, and Sp1 binding motifs by large T-antigen was reduced 2-fold compared to the full length MIEP. Extended truncations diminished trans-activation by large T-antigen. To determine the contribution of a single binding motif in the trans-activation by large T-antigen, a CRE/AP1, an NFkappaB, an Sp1, or a non-consensus Sp1-motif, respectively, was linked to the MIEP TATA-sequence respecting the natural spacing between the two transcription regulatory elements. Only the MIEP TATA-box with the correctly spaced non-consensus Sp1 binding site (GT-motif) was stimulated by large T-antigen. These results suggest that an isolated non-consensus Sp1-motif is important for trans-activation of the MIEP by large T-antigen, but that other cis-acting elements can compensate for this element in the context of the whole MIEP.