The cyst structures of chick ultimobranchial glands were studied by electron microscopy and immunocytochemistry to characterize the type of intermediate-sized filaments present in the cells lining cyst lumina. Electron microscopy showed that the majority of the lumen-bordering cells contained extensive meshworks of intermediate-sized (7-11 nm) filaments, many of which were arranged in bundles. Apical regions of C cells directly bordering on cyst lumina were also filled with thinner (5-6 nm) filaments. Immunoperoxidase staining showed that the majority of cyst epithelial cells were stained intensely with anti-keratin antiserum, but not with anti-neurofilament antiserum, which is a specific marker for neuronal differentiation. The cyst epithelium also showed moderate-to-weak immunoreactivity for actin. Subsequently, the differentiation and maturation of cyst structures related to intermediate filament expression were studied. In 18-day-old chick embryos, keratin immunoreactivity began to appear in the cell clusters destined to form cysts and in the primordial cysts with small cavities. At this time, fine networks of intermediate filaments were already detected in the cells lining the cystic cavities. At 1 day after hatching, the cysts became a consistent feature of ultimobranchial glands. Intermediate filaments associated in bundles were observed, and the intensity of immunostaining for keratins increased. Thereafter, with progressive enlargement of cysts, numbers of intermediate filaments and intensity of keratin immunoreactivity gradually increased with age. Thus, the data indicate that in cyst epithelium keratin filaments are highly organized and may confer the structural strength necessary for cells lining cyst lumina.