Olive (Olea europaea L.) is an economically important crop because of its fruit and oil. Successful olive micropropagation is highly dependence on cultivar, shoot proliferation rate, which is generally low, the rooting of micropropagated olive plantlets is difficult, and the rate of post-transplanting losses is high. In addition, hyperhydricity, a common problem in vitro culture was found to be prevalent. The aim of this study was to establish a micropropagation system for the Turkish O. europaea L. cv. Gemlik. Initially, five different basal media were tested to determine appropriate medium for establishment of in vitro culture and Woody Plant Medium (WPM) was the most efficient. Nodal explants were cultured on WPM containing different plant growth regulators (PGRs) for shoot regeneration. Maximum regeneration frequency and number of shoots per explant were achieved from nodal explants cultured on WPM supplemented with 4.0 mg/L 6-benzyladenine (BA). However, all cultures showed high hyperhydricity and an experimentation was also conducted to resolve the hyperhydricity problem. Hyperhydricity was prevented by changing the gelling agent to Agar-Agar. The shoots regenerated from nodal explants and still attached to initial woody nodal explant were transferred to four different medium formulations each containing 2.0 mg/L zeatin (ZEA) for shoot elongation. Modified Olive Medium (MOM2: OM with three times the concentrations of KNO3) fortified with 2.0 mg/L ZEA was found to be the best for shoot elongation. The elongated shoots were rooted on Olive medium (OM) containing 160 mg/L Putrescine, 1.5 mg/L naphthaleneacetic acid (NAA), 30 g/L mannitol and solidified with 0.65% (w/v) Agar-Agar. Finally, all plantlets were successfully acclimatized in a climate chamber and the plants were transferred to greenhouse conditions. (C) 2019 SAAB. Published by Elsevier B.V. All rights reserved.