Our objective was to obtain parthenogenetic activation of unfertilized human oocytes by puromycin and to try to use this procedure for cytogenetic purposes.The setting was our IVF laboratory.Eighty-two spare oocytes from 34 IVF patients were used. In the first series of experiments 39 unfertilized oocytes were cultured in medium containing 100, 50, or 10 micrograms/ml puromycin for 6 to 24 hr. After the appearance of pronuclei they were transferred to plain medium, further cultured, and cytogenetically analyzed. In the second series of experiments 43 oocytes were cultured for 5 to 10 hr in 10 micrograms/ml puromycin, transferred to plain medium, and fixed for cytogenetic analysis 2 hr after nuclear envelope breakdown.Ninety-one percent of the oocytes in the first experiment showed the presence of one or more nuclei. From the pronucleate oocytes additionally cultured in puromycin-free medium, 46% developed further to the metaphase of the first mitotic division or the two-cell stage and 54% remained arrested at the pronuclear stage. In the second experiment 88% of the treated oocytes showed pronuclei or had cleaved, and after withdrawal from puromycin 96% of the pronucleate oocytes entered mitosis.Puromycin induces haploid as well as diploid parthenogenesis in aged human oocytes. A 5- to 10-hr treatment of oocytes with 10 micrograms/ml puromycin yields the highest percentage of activation, and almost all parthenogenetically activated oocytes enter or develop beyond the first cleavage mitosis. Analysis of mitotic metaphase chromosomes from parthenogenetically activated human oocytes may be a promising new approach to preimplantation cytogenetics.