Open AccessOlive is the most diffused fruit crop in the Mediterranean region, and the improvement of its productivity by conventional and biotechnological approaches is currently required. In vitro cultures have been proven to be very useful for this purpose. In this work, callus cultures of olive (Olea europaea L., cv. Raja) were established from basal tissues of nodal explants and subcultured on OM medium in a growth chamber at 25 °C with a 16h-light photoperiod. Cell clusters with different morphological aspect originated from culture, giving rise to two distinct callus lines that were isolated and long subcultured. Growth and biochemical analyses were then performed to characterize the different proliferation pattern of the two olive callus lines. Compact-type proliferating cultures showed lower growth ability, evaluated by relative growth rate (RGR), than friable-type ones. At biochemical level, differences in pigment, oxidative stress and antioxidative defense marker contents between the two callus lines were observed. In particular, higher chlorophyll and total carotenoid content, higher hydrogen peroxide content and antioxidative enzyme (ascorbate peroxidase, APX, EC 1.11.1.11; glutathione reductase, GR, EC 1.6.4.2; catalase, CAT, EC 1.11.1.6) activities were observed in compact-type proliferating cultures compared to friable ones. On the contrary, at polyamine (PA) level, a higher putrescine (Put) content was detected in the friable-type proliferating cultures. Results evidenced that proliferation of olive callus cells can be characterized by different features involving morphological aspects and biochemical traits associated to cell oxidative condition.