A region located remotely upstream of the human pituitary GH (GH-N) gene and required for efficient GH-N gene expression in the pituitary of transgenic mice was cloned as a 1.6-kb BglII (1.6G) fragment. The 1.6G fragment in the forward or reverse orientation increased −496GH-N promoter activity significantly in pituitary GC and GH3 cells after gene transfer. The 1.6G fragment was also able to stimulate activity from a minimal thymidine kinase (TK) promoter which, unlike −496GH-N, lacked any Pit-1/GHF-1 element. Enhancer activity was localized by deletion analysis to a 203-bp region in the 3′-end of the 1.6G fragment and was characterized by the presence of a diffuse 136-bp nuclease-protected site, observed with pituitary (GC) but not nonpituitary (HeLa) cell nuclear protein. A major low-mobility complex was observed by electrophoretic mobility shift assay (EMSA) with GC cell nuclear protein, and the pattern was distinct from that seen with a HeLa cell extract. The nuclease-protected region contains three A/T-rich Pit-1/GHF-1-like elements, and their disruption, in the context of the 203-bp region fused to the TK promoter, reduced enhancer activity significantly in pituitary cells in culture. A mutation in this region was also shown to decrease enhancer activity in transgenic mice and correlated with a decrease in the 203-bp enhancer region complex observed by EMSA. The participation of Pit-1/GHF-1 in this complex is indicated by competition studies with Pit-1/GHF-1 elements and antibodies, and direct binding of Pit-1/GHF-1 to the A/T-rich sequences was shown by EMSA using recombinant protein. These studies link the A/T-rich sequences to the distal enhancer activity associated with the GH locus control region in vitro and in vivo.