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In previous studies, the distribution of thyrotropes in the chicken pituitary gland has been analyzed by immunohistochemistry using heterologous antibodies. In this study, we examined the distribution of thyroid-stimulating hormone beta subunit-immunopositive (TSHbeta-ip) cells and the expression of TSHbeta mRNA in the pituitary glands of chicken embryos by immunohistochemistry using a specific antiserum to the chicken TSHbeta, in situ hybridization and RT-PCR. Immunohistochemical and morphometric analyses revealed that the TSHbeta-ip cells first appeared on embryonic day 10 (E10) in the pituitary gland and were mainly distributed in the cephalic lobe and that the cell density on E20 was almost 4 times greater than that on E10. The chicken TSHbeta-ip cells could be classified into two types based on morphological characteristics: round-shaped cells and club-shaped cells, which have long cytoplasmic processes. In situ hybridization analysis revealed that TSHbeta mRNA-expressing cells were expressed from E9 in the cephalic lobe and that the extent of TSHbeta mRNA-expressing cells coincided with that of TSHbeta-ip cells. RT-PCR also showed that TSHbeta mRNA was expressed from E9 and that Pit-1 mRNA was expressed from E5. These results clearly demonstrated that the expression of chicken TSHbeta mRNA starts on E9, that TSHbeta-ip cells appear on E10, mainly in the cephalic lobe, and that TSHbeta-ip cells can be classified into two cell types (round- and club-shaped cells)., (Copyright 2004 S. Karger AG, Basel)