Viable egg cells of Datura stramonium (Solanaceae) were isolated using enzymatic digestion and mechanical dissection. The three cells comprising the egg apparatus (the egg cell and two synergids) were removed from the female gametophyte by altering the micropylar position with a dissection needle. Generally, the egg apparatus cells were readily released from the cut end of the ovule. In order to isolate the egg cells, ovules were placed in an isolating solution containing 0.04% CaCl₂, 1% BSA, and 12% mannitol, seven egg apparati were isolated from 40 ovules within one hour period. However, in some cases, it was difficult to separate the egg cells from the two synergids. Separation of the egg and synergid cells required incubating the ovules in an enzymatic solution containing 1% pectinase (Serva), 1% cellulase (Onozuka RS), and 12% mannitol for 30 min, and transferring them to the above-mentioned isolating solution. Following this protocol, eight egg cells were isolated from 40 ovules within two hours, and the egg cells were easily separated from the two synergids. Subsequently, isolated egg cells were used for in vitro fertilization to explore the mechanisms of egg development and fertilization.