Abstract The role of βAPP gene transcription and promoter regulation in modifying amyloid β-peptide (Aβ) levels is not well understood. Increased production of Aβ or changes in Aβ 42/Aβ 40 ratio by fibroblasts occurs in the presence of mutant presenilin or βAPP alleles in familial Alzheimer's disease subjects. Both βAPP mRNA and Aβ levels are increased in trisomy 21. The APP gene promoter is in a class of housekeeping genes and contains two putative consensus sites for the binding of transcription factor AP1. Electrophoretic mobility shift (EMSA) and DNase protection assays using human fibroblast and HeLa nuclear extract identified specific protein binding with novel Sp1-like properties to both a near-upstream and a downstream domain of the βAPP promoter. The upstream binding activity was localized to a putative AP1 consensus site and its immediate 5′-adjacent GC-rich element. However, c-Jun antibody and competition experiments had no effect on binding to this domain. A series of 5′-deleted βAPP promoter–reporter gene transfections in HeLa and fibroblast cells showed that the domain-containing region, n.t. −383 to −348, exerts a 2.9-fold activating influence on basal pβAPP-reporter transcription. When subcloned to test enhancer function, the 5′-GC element/‘AP1 site’ tandem construct conferred four-fold greater activity than either element alone and two-fold greater than the more 3′-situated HSE consensus sequence. Phorbol ester treatment had no effect in these reporter assays. This element shares homology and binding properties with a domain immediately 5′ to the downstream E-box/USF element. An interaction model involving both domains and looping of interjacent DNA is proposed. We conclude that this newly described binding protein–enhancer complex is required for full βAPP promoter activation.