The pituitary hormones LH, FSH, and TSH are heterodimers composed of a common alpha-subunit and unique beta-subunits. We demonstrate that 4.6, 2.7, 1.49 or 0.48 kilobases (kb) mouse alpha-subunit 5'-flanking sequences are sufficient for transgene expression in both gonadotropes and thyrotropes but not in inappropriate pituitary cells. In contrast, transgenes with bovine or human alpha-subunit flanking sequences have been shown to confer reporter gene expression only to gonadotrope cells, suggesting that the elements regulating cell-specific expression may differ between species. Equal levels of reporter gene expression were conferred by 5.0 and 0.48 kb in transiently transfected thyrotrope tumor-derived cells. In contrast, in transgenic mice, high level expression was only obtained with 4.6 kb 5'-flanking sequences, indicating the presence of an enhancer element between 4.6 and 2.7 kb. The 4.6 kb of 5'-flanking sequences are sufficient for both hormonal and developmental regulation of transgene expression. Mice rendered hypothyroid by radiothyroidectomy had significantly higher levels of transgene expression than either hyperthyroid or euthyroid animals. The temporal and spatial pattern of transgene expression in Rathke's pouch paralleled that of the endogenous gene; the onset of transgene expression occurred by embryonic day 9.5. Low level expression of both the transgene and the endogenous alpha-subunit gene were detected in some unexpected peripheral sites, such as the embryonic extraocular and olfactory regions, suggesting that alpha-subunit may have a more diverse role in development than previously considered.