Purpose: In this prospective randomized study the effectsof enzymatic treatment of zona pellucida of blastocysts onimplantation and pregnancy rates were evaluated in a groupof patients who had more than five embryos on day 3. Methods: Forty-six patients with a mean age of 29.8 ± 4.5years and mean duration of infertility of 6.72 ± 0.63 yearshad blastocyst stage transfers, with a mean number of2.9 ± 0.1 embryos replaced per patient. Patients were randomlydivided into two groups. The first group consisted ofpatients (n = 22) who had zona intact blastocyst stagetransfers and the second group consisted of patients (n = 24)who had zona manipulated (enzymatic treatment) blastocyststage transfers. Patient and cycle characteristics were similarin both groups. A commercial cell and serum-free sequentialculture system was used for all embryos. Results: Overall blastocyst formation rate was 50.3%.Transfer could be done in all patients. The positive β-humanchorionic gonadotropin rate in the zona intact group was50% (11/22) and in the zona-manipulated group was 70.8%(17/24). Clinical pregnancy, ongoing pregnancy, andimplantation rates in zona intact and manipulated groupswere 45.5%, 27.3%, and 19%, and 62.5%, 45.8%, and 24%respectively. Although implantation and pregnancy rates inthe zona-manipulated group were higher, there were nostatistically significant differences in terms of these variablesbetween two groups. No triplet pregnancy was obtained ineither group, and the twin pregnancy rate was 20% (2/10)in the zona intact group and 13.3% (2/15) in thezonamanipulated group. Conclusions: With further improvements in the embryoculture systems it will become possible in the near future toachieve high implantation rates even with single blastocysttransfers. Enzymatic treatment of the zona pellucida seemsthat it does not alter the pregnancy and implantation rates,but further studies with larger group of patients are neededto clarify the real effect of this zona manipulation onpregnancy outcome.