GPAT and AIRC encode enzymes for steps one and six plus seven respectively in the pathway for de novo purine nucleotide synthesis in vertebrates. The human GPAT and AIRC genes are divergently transcribed from a 558 bp intergenic promoter region. Cis-acting sites and transcription factors important for bidirectional expression were identified. A cluster of sites between nt 215 and 260 are essential, although not sufficient, for expression of both genes. Two proteins from HepG2 cell nuclear extract, identified as NRF-1 and Sp1, bound to the promoter at sites within the 215-260 region. NRF-1 was required for stable binding of Sp1. Deletion of a 5'promoter region including nt 215-260 resulted in decreased expression of GPAT and AIRC in transfected HepG2 cells. The decreased expression was accounted for by point mutations in an NRF-1 site and either of two flanking sites for Sp1. These transcription factors account in part for the coordinated expression of human GPAT and AIRC.