Although there is evidence that the LIM homeodomain transcription factor, Lhx2, can stimulate transcription of the glycoprotein hormone -subunit gene, the role of Lhx2 in regulating TSH -subunit has not been established. In the present studies, the ability of Lhx2 to regulate transcription of the TSH -subunit gene has been examined. In the thyrotrope-derived TT1 cell line, Lhx2 expression was found to be induced by treatment with either TRH or cAMP, consistent with the possibility that Lhx2 may play a role in mediating the ability of this signaling pathway to stimulate TSH gene expression. Transient, forced overexpression of Lhx2 stimulated activity of a TSH -subunit reporter gene. Deletion studies provided evidence that the -177 to -79 region of the TSH -subunit promoter was necessary for stimulation of reporter gene activity by Lhx2. A gel mobility shift assay provided the evidence that Lhx2 can bind to this region of DNA. DNase I footprinting studies demonstrated that two distinct regions of the TSH promoter, -118 to -108 and -86 to -68, are protected by Lhx2 from nuclease digestion. These regions contain repeats of the sequence, 5'-(G/T)CAAT(T/A)-3'. Mutation of this sequence, especially in the -86 to -68 region substantially decreased Lhx2 responsiveness of the TSH -subunit reporter gene. In addition, a DNA fragment containing the -177 to -79 region of the TSH promoter was found to confer Lhx2-responsiveness to a minimal promoter. These results provide multiple lines of evidence consistent with a role for Lhx2 in modulating expression of the TSH -subunit gene.