The molecular organization of cellulose I-beta microfibrils in the tunic of Halocynthia papillosa was analyzed by high-resolution cryoelectron microscopy on ultrathin cross sections of artificially highly oriented microfibrils. The arrangement of cellulose chains intersected by the 0.6-, 0.53-, and 0.39-nm equatorial lattice planes was clearly imaged over the whole area of a parallelogram shaped cross section of a microfibril. One edge of the parallelogram was parallel to the 0.6-nm lattice plane, while the other did not correspond to a crystallographic plane. Such organization is distinct from previous findings on algal cellulose I-alpha-rich microfibrils, which have an almost square cross section bounded by both 0.6- and 0.53-nm crystallographic planes. A tentative model for microfibril formation is proposed by introducing a two-step biocrystallization mechanism: the formation of molecular sheets spaced by 0.53 nm between adjacent molecules, followed by self-deposition of these sheets by hydrogen bonding between them.