The effect of long-term treatment (52 weeks) with high doses of 17β-estradiol (1.28 mg/kg/week intramuscularly) on gonadotrophs was studied in the pituitary gland of the beagle bitch. For immunochemical staining the immunoperoxidase technique and antisera to the specific beta (β) subunits of FSH and LH were employed. For control purposes antisera to the following hormones were also used: bovine TSHβ, canine GH, canine PRL and porcine ACTH1. In the pars distalis and pars tuberalis of control bitches, in addition to the cells which react solely with antisera to either LHβ or FSHβ, most cells were reactive to both antisera. The cells stained for FSHβ were less numerous than those shown to contain LHβ. TSHβ, PRL, GH and ACTH/MSH were localized in distinctly different cell types in the pars distalis of all control animals. In the treated bitches, almost complete regression of cells classically identified as gonadotrophs and stained for LHβ was observed. On the other hand, using the antiserum to FSHβ, selective immunochemical staining was localized in cells fitting the morphological characteristics of TSH cells. All these cells were also stained for TSHβ. However, a few cells were also shown to react solely with the antiserum to TSHβ. These cells, which seem to contain both TSHβ and FSHβ, were further clearly differentiated from PRL, GH and ACTH/MSH cells on the basis of their cytological features, intraglandular distribution and by immunochemical double staining. These observations support the concept that the one cell-one hormone theory may not necessarily apply to the glycoprotein hormones of the dog pituitary gland.