To clarify the involvement of actin in the formation of the yeast cell wall, reverting protoplasts of Schizosaccharomyces pombe were used as a simple model system. Actin of reverting protoplasts was labeled with rhodamine-conjugated phalloidin and observed by conventional fluorescence microscopy and laser scanning confocal microscopy. A close spatial as well as temporal relationship between actin and cell wall formation was observed in protoplast reversion. That is, the site of actin 'dots' in the reverting protoplasts coincided with the site of new wall formation and the timing of rearrangement of actin coincided with the initiation of cell wall formation and with the timing of cell wall expansion. Treatment of reverting protoplasts with cytochalasin D (CD) further clarified the close relationship between actin and cell wall organization. The effect of CD was dose dependent. A high dose of CD caused the absence of actin as well as the complete inhibition of cell wall formation. A low dose of CD caused weakly stained unlocalized actin, which induced grossly aberrant cell wall deposition as well as substantial changes in the morphology of the reverting protoplasts. These results demonstrated that actin is associated with initiation of cell wall formation, the proper deposition of cell wall materials, and maintaining the normal morphology of reverting protoplasts. Scanning electron microscopy revealed the presence of a fibrillar net structure on the surface of non-treated control reverting protoplasts. However, the absence of a fibrillar network on the surface of reverting protoplasts was observed in the presence of a high concentration of CD. Lack of localization of microfibrils as well as poor development of the fibrillar network were also observed in the presence of a low concentration of CD. Recovery experiments confirmed the close relationship between actin and cell wall formation.