This study asks whether retinal adhesiveness is affected by cytochalasin D, a drug that is known to alter the apical morphology of the retinal pigment epithelium (RPE). Cytochalasin D was injected intravitreally in Dutch rabbits and retinal adhesiveness measured 0.5 to 72 h later by two methods: in vitro peeling of the retina from retinal pigment epithelium to observe the amount of adherent pigment, and in vivo measurement of the pressure needed to achieve retinal separation. Electroretinograms were recorded, and RPE apical morphology was examined by scanning electron microscopy. The injection of 60 microM cytochalasin D caused in vitro retinal adhesiveness to fall within 3 h to 10% of normal although the electroretinogram (a, b, and c-waves) remained normal. Smaller doses of cytochalasin D had a lesser effect. The RPE apical surface at 3 h showed large bullet-like microvilli, swollen cone sheaths, and an absence of filamentous microvilli. The severity of these changes was dose-related. At 72 h after cytochalasin D, retinal adhesiveness had largely recovered, and RPE apical morphology appeared normal again. Thus, cytochalasin D weakens retinal adhesiveness acutely but reversibly, and both the initial effect and recovery correlate with changes in RPE microvillar morphology. This suggests that actin microfilaments may be involved in mechanisms of retinal adhesion.