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Os objetivos deste trabalho foram estabelecer in vitro as cultivares de oliveira 'Arbequina' e 'Maria da Fé' e avaliar a influência das tampas com membranas permeáveis a gases na morfogênese in vitro dessas cultivares. Inocularam-se segmentos nodais com gemas previamente descontaminadas pelo protocolo aqui desenvolvido. Utilizaram-se o delineamento inteiramente casualizado (DIC) em esquema fatorial 2³, duas cultivares; dois meios de cultura OM (Olive medium) (OM + 20 µM de zeatina [1]; e OM + 20 µM de zeatina + 10 µM de GA3 [2]); dois tipos de vedação (tampa rígida sem orifício e com membrana porosa) com cinco repetições/ tratamento; e a unidade experimental constituída por quatro tubos de ensaio. Avaliaram-se: a porcentagem de contaminação total; a porcentagem de contaminação fúngica e bacteriana; o número de gemas intumescidas; o número de brotos; e a porcentagem de oxidação. Aos 30 dias de cultivo, constatou-se a contaminação de 15% e 8,8% dos explantes de 'Arbequina' e 'Maria da Fé', respectivamente. Em 'Arbequina', 33,3% e 66,7% ocorreram por contaminação fúngica e bacteriana, respectivamente. Em 'Maria da Fé', 28,6% e 71,4% decorreram de contaminação fúngica e bacteriana, respectivamente. O número de gemas foi superior (pThe objectives of this work were to establish cultivars of Olea europaea L., namely 'Arbequina' and 'Maria da Fé', and to evaluate the influence of gas exchange on in vitro morphogenesis of these cultivars. Under aseptic conditions, single-bud nodal segments were previously decontaminated using disinfestation protocol developed in this work. The treatments followed a completely randomized design, in a 2³ factorial scheme: two cultivars and two olive (Olive medium) culture media (OM) (OM + 20 µ M zeatin [1]; and OM + 20 µ M zeatin + 10 µM GA3 [2]); and two types of sealing (rigid lid without orifice and porous membrane) in five repetitions/treatment. The experimental unit consisted of four tubes. The following parameters were assessed: percentage of total contamination; percentage of fungal and bacterial contamination; number of explants with swollen buds; number of axillary buds, and oxidation percentages. At 30 days of cultivation, the total contamination indexes of 15% and 8.8% were recorded for 'Arbequina' and 'Maria da Fé', respectively. For bacterial and fungal contamination percentages, respectively, 'Arbequina' had 33.3% and 66.7%, whereas 'Maria da Fé' presented 71.4% and 28.6%. Regarding the number of responsive cultures showing swollen buds, there was no difference between cultivars when rigid lids without porous membranes were used. However, lids with porous membranes led to statistically superior values for 'Arbequina' as compared to 'Maria da Fé'. For the number of shoots, culture medium 1 was statistically superior to 2. There was no statistical difference for oxidation percentages among treatments. Thus, because of the low level of contamination, it is suggested to use the disinfestation protocol developed in this work. In addition, the use of culture medium 1 and lids with porous membranes did favoured further development of axillary buds into plants.