The present studies were performed to establish the effects of the size and number of artificial holes produced in the zona pellucida (ZP) on hatching and trophoblast outgrowth in vitro. Limited partial zona dissection (PZD) produced small, narrow incisions, and zona drilling with acidic Tyrode's (AT) across a larger area in the ZP was used to produce bigger round holes. Some embryos were micromanipulated once; others were micromanipulated several times. Blastocysts hatched preferentially through the artificial gaps, but completion of hatching was dependent on the size of the hole. Only 16% (26/167) of PZD embryos migrating through narrow holes hatched completely; the remainder were trapped in a typical figure-eight shape. Seventy-two percent (43/60) of those migrating through larger PZD holes hatched, but trophoblast outgrowth was not observed. Significantly (P less than 0.001) more AT-blastocysts hatched (248/270; 92%) and showed trophoblast outgrowth (176/248; 70%). Simultaneous hatching through several openings was rarely observed in AT-embryos (14/167; 8%), but this did occur in 36% (73/201) of the PZD embryos. Trapping of PZD-embryos could be almost entirely avoided by drilling with AT elsewhere on the ZP. Embryos with multiple holes in their zonae preferentially hatched through the largest opening. The results suggest that the ability of microsurgically treated human embryos to fully hatch in vitro, should be carefully (re)assessed prior to application of clinical micromanipulation systems. Micromanipulated embryos with small holes in their zonae may be rescued by performing an additional more aggressive opening procedure elsewhere on the ZP.