In the present study the in vitro propagation from adult native plants of Juniperus oxycedrus was investigated. The effect of seasonal explant collection (spring, summer, winter), in varying media and cytokinin type were examined. The decontamination process of explants taken in the summer was more efficient than that of the other seasons (57.3% of the explants survived). In the spring a low performance in shoot formation took place both on hormone free Murashige and Skoog (MS) media and Rugini Olive Medium (OM) or each containing 6-benzyladenine (BA) at 1.0 mg L-1 by which shoots did not elongate more than 0.5 cm. In the summer OM was used, and a higher in vitro performance was observed on medium containing 0.1 mg L-1 6-(γ,γ-Dimethylallylamino)purine (2iP); the shoot formation percentage was 72.0%, and respectively 3.1 shoots, 1.4 cm in length, were formed. In the winter, the shoot formation percentage was 92.0-100.0%, and respectively 3.9 shoots of 0.4 cm length were formed on medium containing 1.0 mg L-1 2iP. The multiplication phase on cytokinin-containing media led to a satisfactory proliferation rate in terms of shoot formation percentage and shoot number. The higher shoot number (3.3) was recorded on media containing 2iP at 1.0 mg L-1, while the shoot length was longer (2.0) cm, on hormone-free media. Initiation of roots was not observed during the rooting phase on hormone-free half-strength OM, or supplemented with 1.0 and 2.0 mg L-1 IBA. Spontaneous rooting at a low percentage (10.0%) was observed on OM media supplemented with 0.1 mg L-1 2iP and successful acclimatisation took place on a 1:1 (v/v) ratio of peat to perlite substrate.