Our objective was to induce enucleation (IE) of activated mouse oocytes to yield cytoplasts capable of supporting development following nuclear transfer. Fluorescence microscopy for microtubules, microfilaments, and DNA was used to evaluate meiotic resumption after ethanol activation and the effect of subsequent transient treatments with 0.4 μg/ml of demecolcine. Using oocytes from B6D2F1 (C57BL/6 × DBA/2) donors, the success of IE of chromatin into polar bodies (PBs) was dependent on the duration of demecolcine treatment and the time that such treatment was initiated after activation. Similarly, variations in demecolcine treatment altered the proportions of oocytes exhibiting a reversible compartmentalization of chromatin into PBs. Treatment for 15 min begun immediately after activation yielded an optimized IE rate of 21% (n = 80) when oocytes were evaluated after overnight recovery in culture. With this protocol, 30–50% of oocytes were routinely scored as compartmentalized when assessed 90 min postactivation. No oocytes could be scored as such following overnight recovery, with 66% of treated oocytes cleaving to the 2-cell stage (n = 80). Activated cytoplasts were prepared by mechanical removal of PBs from oocytes whose chromatin had undergone IE or compartmentalization. These cytoplasts were compared with mechanically enucleated, metaphase (M) II cytoplasts whose activation was delayed in nuclear transfer experiments using HM-1 embryonic stem cells. Using oocytes from either B6D2F1 or B6CBAF1 (C57BL/6 × CBA) donors, the in vitro development of cloned embryos using activated cytoplasts was consistently inferior to that observed using MII cytoplasts. Live offspring were derived from both oocyte strains using the latter, whereas a single living mouse was cloned from activated B6CBAF1 cytoplasts.