Eggs isolated from animals spawned with 10(-3) M serotonin were inseminated with sperm concentrations ranging from 10(3)-10(6) sperm/ml. Multiple sperm attached to the surface of the egg and sperm incorporation occurred within 3 min postinsemination (PI). Sperm mitochondria, centrioles, and flagellum were also incorporated. Incorporation was essentially complete by 6 min PI. In the egg cortex, the sperm head rotated 180 degrees, and a rapid translocation of the sperm through the cytoplasm towards the egg interior began by 5-6 min PI. In heavily polyspermic inseminations, translocations of the sperm were either minimal or nonexistent. In monospermic eggs, nuclear decondensation occurred after translocation was complete, beginning by 9-10 min PI. A male pronucleus began to develop in the cytoplasm by 21 min PI and enlarged to 20 microns before fusing with the female pronucleus. Oscillation of the egg cytoplasm and mitotic spindle apparatus was observed immediately prior to cleavage. Cleavage occurred at 60 min PI. Sperm incorporation and pronuclear formation were confirmed with fluorescent and confocal microscopy using the DNA-specific dyes Hoescht 33342 and 7-aminoactinomycin D. In sperm concentrations > 10(4) sperm/ml, 26-76% of the eggs exhibited polyspermy. The high incidence of polyspermy suggests that rapid, effective blocks to polyspermy were not present or were ineffective in a significant proportion of serotonin-spawned eggs.