Detailed experimental procedures are described for the preparation of thin-walled giant phosphatidylcholine vesicles, which are useful for microinjections. In these microinjection experiments, a target vesicle (typically about 50 to 100 mu m in diameter) was punctured by a microneedle and an aqueous solution was injected into the internal volume of the vesicle. The method, which was used for giant vesicle preparation, is a modification of the so-called electroformation method, originally described by Angelova and Dimitrov (Faraday Discuss. Chem. Sec. 1988, 81, 303-311 and 345-349). With this method, the vesicles grow in an investigation chamber at a platinum wire in an aqueous medium with the help of an alternating electric field, and we have investigated how the experimental parameters (in particular applied voltage and frequency and ionic strength of the aqueous medium) influence the vesicle formation process. Using a specially constructed investigation chamber and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) as lipid, the applied voltage was varied between 0.6 and 10 V, holding the frequency constant at 10 Hz. At voltages <5 V, the giant vesicles formed often appeared under the microscope as nonspherical ("cut spheres") and open, "mushroom-like" structures. Often, however, nonsphericity was only an optical artifact, and closed vesicles could be distinguished from open structures by microinjecting fluorescent dye molecules, which in the case of an open structure immediately leaked out. At voltages >5 V, closed structures were observed. At constant voltage (1.3 V), "cut spheres" and "mushroom" type structures appeared mainly in the frequency range 10-100 Hz. Between 0.2 and 2 Hz, mainly closed structures were formed. Typical conditions for vesicle formation useful for microinjections were 2 V and 10 Hz.