Expression of the TSH beta subunit gene is restricted to the thyrotroph cells of the anterior pituitary. Previously we identified several AT-rich DNA elements within the murine (m) TSH beta 5'-flanking region, denoted as D1 (-253 to -227), P4 (-142 to -131), P3 (-126 to -112), P2 (-106 to -98), and P1 (-76 to -68) which bind thyrotroph-specific factor(s). These sites are related to, but distinct from GHF-1 and LSF-1 binding sites, which restrict GH and PRL gene expression to pituitary somatotrophs and lactotrophs, respectively. To determine whether different pituitary cell types contain related factors capable of activating the mTSH beta promoter, cell-free transcription studies were performed using extracts from GH4 rat pituitary somatomammotroph cells. AI-through the endogenous mTSH beta gene is not expressed in GH4 cells, in vitro transcription of the mTSH beta promoter, normalized to the Rous sarcoma virus internal control, revealed faithful transcription initiation from the authentic mTSH beta CAP sites in GH4 but not in HeLa cell extracts. Cell-free transcription analysis of mTSH beta 5'-deletion mutants revealed consistent promoter activity with deletion to position -46 but complete loss of activity when deleted to position -9. To better define the specific factors in pituitary somatomammotrophs which interact with and activate the mTSH beta promoter, DNase I protection and gel-shift studies were performed using extracts from GC rat pituitary somatomammotroph cells and DNA affinity-purified lactotroph-specific transcription factor, LSF-1, required for rat PRL promoter activity, and purified from GC cells. These cells contain a factor(s) which binds to thyrotroph-specific elements of the mTSH beta promoter. These studies also show that LSF-1 binds the D1 and proximal thyrotroph-specific elements of the mTSH beta promoter and is capable of reconstituting the trans-activation of the mTSH beta promoter in HeLa nonpituitary cell extracts in vitro. Conversely, nuclear factors present in TtT-97 murine thyrotrophs bind the proximal lactotroph-specific elements on the rPRL promoter. This in vitro transcription assay provides a means to biochemically dissect the trans-activation of the mTSH beta promoter and to determine the functional overlap of distinct pituitary cell-specific factors in regulating GH, PRL, and TSH beta gene expression.