Increasing evidence suggests that the hypophyseal pars tuberalis (PT) plays a key role in the transduction of light/dark (melatonin) information to the endocrine system. It has been shown that PT-specific cells express melatonin receptors and thyrotropin hormone (TSH) subunits. However, these cells do not resemble thyrotrophs or any other of the pars distalis (PD) cells. There is evidence that PT-specific cells secrete a glycoprotein hormone designated as ‘tuberalin’. We have identified a putative tuberalin of 21 kDa (tuberalin II) and have raised antibodies against it. To further investigate whether tuberalin II is a distinct secretory compound of the PT, absorption studies of antituberalin II with TSH or with an extract of the rat PD containing β-TSH, β-luteinizing hormone (LH) and the common α-subunit of glycoprotein hormones (GSU), were performed. Neither of the absorption tests abolished the immunoreactivity of the PT to antituberalin II, suggesting that tuberalin II is different from TSH or the other PD glycoprotein hormones. Double immunofluorescence analyses using antibodies against tuberalin II, β-TSH and GSU revealed that in the developing and adult PT there are 3 populations of PT-specific cells expressing tuberalin II and GSU (type 1), β-TSH and GSU (type 2) and tuberalin II, β-TSH and GSU (type 3). This further indicates that tuberalin II and β-TSH correspond to different compounds and that they may be expressed either by different cells types or coexpressed in a 3rd cell type. The distribution and temporal expression of tuberalin II, β-TSH, β-LH and GSU were investigated in the developing pituitary gland. At E14.5, tuberalin II and GSU were expressed by cells of the PT primordium but not by the PD and pars intermedia primordia. The onset of expression of β-TSH, β-LH and GSU in cells of the PD occurred about 1 day later, further indicating the distinct nature of tuberalin II and supporting the earlier view that the secretion of polypeptides from the fetal rat pituitary gland begins in PT-specific cells.