The Olive (Olea europaea sativa L.) plays an important role in the economies of countries in the Mediterranean area. The in vitro culture of Olive, a particular difficult species to propagate in vitro, required the development of a specific medium formulation. Rugini medium is dedicated to the proliferation of the olive shoots. The medium has an enriched composition compared to MS. Olive tissues are characterized by a high content of Ca, Mg, S, Cu and Zn. The best nitrogen source is a combination of NO3- and NH4+ supplemented with glutamine 2,19 mg/l. The better carbon source is mannitol (30-36 gr/l) compared to sucrose. A better cytokinin to be used is zeatin: 1 mg/l if filter sterilized, 3-4 mg/l when autoclaved. TDZ and 2iP are less effective. Shoots grow more rapidly compared to other media. The proliferation rate increases and more tender, sturdier shoots with less basal callus are obtained.