Abstract: An efficient protocol was developed for the establishment of aseptic conditions, as a pre-requisite for meristem tip culture of olive (Olea europa L.) CV. Pantaloon. Shoot tips 0.5-0.8mm in length were used as explants source from the field-grown olive plants. Before cultured on Olive Initial Medium (OIM) for shoot proliferation and callus induction, these shoots were disinfected with four different disinfection procedures. Sterile and growing cultures were obtained, only from explants sterilized with 2.5mm HgCl2 for 3 min followed by 50% Chlorox for 15min. Sterile explants responded to the growing media either by developing into leafy shoots or by producing callus. This paper outlines the aseptic protocols for obtaining the sterile explants as the starting material to initiate in vitro cultures in olive CV. Pantaloon.