In order to study the expression of the morphological and functional characteristics of human thyroid cells, 3-dimensional cultures were carried out in collagen gel. This substrate allows the cells to retain their organization in follicles with a normal polarity. Cellular polarities appeared normal at the time of collagen embedding, but there was a delay of 4-5 days in culture before the maximal TSH stimulation of 125I- uptake and of cAMP accumulation occurred. In normal and adenoma-derived cells, 125I- uptake, which could be increased by TSH, was demonstrated. cAMP accumulated in the culture medium and thyroglobulin was secreted into the follicle lumen. Of the 4 differentiated carcinomas for which the 72-hr uptake of 125I- was measured, only 2 displayed slight 125I- uptake and response to TSH. Thus, human thyroid cells exhibit better morphological and functional differentiation in collagen gel culture than in monolayer culture. Furthermore, in a variety of pathological cases studied, the expression of specific characteristics in culture varied in a fashion similar to differences observed in vivo.