We have developed a culture technique by which the normal configuration of follicles and most other structural characteristics of the thyroid gland are well preserved. Small tissue fragments (diameter 0.5 to 0.9 mm) from various mammalian species were prepared and kept in hydrophobic culture dishes to prevent their attachment. It was essential to raise the oxygen concentration in the incubator to 50% and the D-glucose concentration to 5.6 mM. Under these conditions, mini organs were formed by the outgrowth of epithelial cells from follicles located on the fragment surface and opened during tissue preparation. After 2 to 3 days, the mini organs were enclosed by a confluent and tight monolayer of follicle cells and [3H]thymidine incorporation returned to background levels. The structural integrity of follicles underneath the monolayer and in the center of the mini organs could be maintained for at least several weeks. Light microscope autoradiographs of mini organs incubated with iodide, sulfate, or phosphate revealed that the vectorial transport and the posttranslational modifications of thyroglobulin were maintained. An assay for the precise quantitation of biosynthetic activities in individual mini organs was developed. The observations indicate that thyroid-specific functions in mini organs can be preserved for prolonged periods of time.