Previous work revealed that, in the beta chitin secretion gland of the hydrothermal vent worm Riftia pachyptila, cup-shaped microvilli (cups) were in close contact with a chitin-like microfibril. A model was suggested, involving a transmembrane synthesis of chitin microfibrils. The aim of the present work is to localize chitin at a cellular level, in the case of animals actively secreting their chitinous tube. Thus, freshly sampled animals were repressurized after in situ collection (2600 m depth). Tube secretory activity was demonstrated during the time the animals were kept in the pressure vessels. Chitin localization was carried out on thin sections of the glands, by means of diffraction contrast transmission electron microscopy and wheat germ agglutinin/gold conjugate labeling. Chitin was unambiguously evidenced inside the cavity of the cups, therefore proving the chitinous nature of the anchored microfibril. In addition, both techniques failed to localize chitin in the intracellular compartment, especially around vesicles associated to the cups. It is concluded that the cup is the site of assembly of R. pachyptila's giant beta chitin microfibrils. Last, the in vitro secretory activity is an indication of good physiological state of the repressurized animals, despite the drastic environmental changes that occur during collection. (C) 1995 Academic Press, Inc.