The ploidy of single-pronucleated human zygotes obtained after conventional in vitro fertilization was determined by fluorescent in situ hybridization (FISH) using multiple simultaneous probes for gonosomes and autosomes. After zona removal the single-pronucleated zygotes were exposed to cytochalasin B, and the pronucleus, surrounded by scant cytoplasm and the plasma membrane (karyoplast), was divided from the rest of the egg (cytoplast). The karyoplasts and the corresponding cytoplasts were analyzed separately by FISH. Of the 16 zygotes analyzed, 10 had haploid pronuclei and 6 were diploid. Four diploid pronuclei contained XY chromosomes, and 2 contained XX chromosomes. These results suggest that during the course of their interaction, human gamete nuclei can associate together and form diploid, single-pronucleated zygotes. These findings confirm a newly recognized variation of human pronuclear interaction during syngamy.