The dynamics of cell wall regeneration in Candida albicans protoplasts was investigated by fluorescence microscopy using a new fluorescent dye, Fungiflora Y, which specifically binds to beta-linked polysaccharides, as well as by electron microscopy. When freshly prepared protoplasts were incubated in osmotically stabilized medium, multiple binding sites of Fungiflora Y were detected on their surface within 15 min of incubation. At this initial stage of reversion, protoplasts began to form microfibrils outside the cytoplasmic membrane. From 1 h onward, the whole cell surface displayed a bright fluorescence. After 3 h of incubation, the reverting protoplasts were covered by an interwoven network of microfibrils. After 5 h of incubation, although most of the revertants had become osmotically resistant, regenerated cell walls in which the constituent materials appeared highly integrated were observed to be still monolayered in profile. Cell wall regeneration was complete after 24 h of incubation, at which time the reverted cells were capable of proliferating.