The effects of cytochalasin B (CB), which acts on microfilaments, on sperm penetration into eggs of a teleost fish were investigated ultrastructurally. Eggs from the rose bitterling, Rhodeus ocellatus ocellatus, were pre-treated in physiological saline containing CB and inseminated in water also containing CB. Microvilli at the sperm entry site (SES) under the micropyle disappeared or shortened in length following CB treatment. However, a spermatozoon attached to and fused with the SES of CB-treated eggs. The spermatozoon present in a swollen mass (SM) remained at the egg surface even after membrane fusion and did not enter the cortex. It was unclear whether or not sperm movement from the cortex to the inner cytoplasm is CB-sensitive. The SM formed and plugged the micropyle. CB did not inhibit cortical alveolus breakdown. Based on the present experiments with fish eggs, it is concluded that CB inhibits sperm movement from the egg surface to the cortex, but not sperm attachment (binding), membrane fusion and SM formation during the process of sperm penetration.