Rat anterior pituitary cells were dissociated and subjected to 4 h of unit gravity sedimentation. Eighty-five 8-ml fractions were collected, and nine pooled fractions were placed in monolayer culture for 1 week. The attached cells were immunocytochemically stained for TSH using an antiserum previously shown to be specific for the beta subunit of TSH and all culture media saved for subsequent assay of TSH. Thyrotrophs were localized both immunocytochemically and by radioimmunoassay to 1-2 fractions from the top of the sedimentation chamber. Typically, it was found that 60-80% of the cells in these fractions were immunocytochemically identified as thyrotrophs. Electron microscopic observations indicated that such cells displayed the classical morphological features associated with thyrotrophs. The enriched thyrotroph cultures responded to 1.0 mM dibutyryl cyclic AMP with an increased release of TSH indicating that the intracellular secretory machinery was not altered by the procedures employed. In all cases, the basal TSH secretion decreased with time in culture. In addition, the fractions containing the most TSH in the culture media were usually one fraction lighter (lower sedimentation rate) than the fractions which were found immunocytochemically to contain the highest percentage of thyrotrophs. The results suggest the possibility of two functionally distinct thyrotroph cell types as has been suggested for the pituitary somatotrophs.