The pS2 gene is estrogen responsive in hepatocarcinoma cells (HepG2) in the presence of estrogen receptor alpha (ERalpha). The estrogenic activity is mediated through an estrogen response element (ERE) in the 5'-flanking region of the pS2 gene; however, an activator protein 1 (AP1) response element located close to the ERE in the pS2 promoter has also proven essential for a maximum response to estrogen. In the present study, we show estrogen-induced synergistic activity by the p160 coactivator steroid receptor coactivator-1 (SRC-1), mediated via the ERE and the AP1 response element in the pS2 promoter. In addition, we present data that support an interaction between the ERE and the AP1 motif via SRC-1. The related but distinct p160 coactivator, transcriptional intermediary factor-2, was a more potent activator of pS2 gene expression. In addition, transcriptional intermediary factor-2 was less dependent on an intact AP1 response element in the pS2 promoter than SRC-1. Furthermore, the type of ERE in the pS2 promoter influenced the potentiation by SRC-1, supported by less dependence on the AP1 motif when the natural ERE was substituted for by a consensus ERE. These results highlight several mechanisms whereby fine-tuning of estrogen responsiveness of an individual gene may be achieved.