We studied the self-incompatibility of two main Spanish olive (Olea europaea L.) cultivars, `Picual' and `Arbequina', by testing the selfing of the seeds with microsatellites. For this purpose, we used a rapid single-seed DNA extraction method and four highly polymorphic microsatellites. We analyzed seeds produced in branches bagged for selfing from mono- and multi-cultivar orchards in 2002 and 2003. We did not find any seed coming from selfing in the bagged branches, for either cultivar, in the two types of orchards. Additionally, we tested seeds coming from free pollination in mono-cultivar orchards from different locations. In the case of `Picual' olive, only three seeds out of the 70 collected were the product of selfing, although they came from mono-cultivar orchards located in areas where the cultivar used as the female parent was predominant. From the 20 seeds of `Arbequina' olive harvested in the middle of two high-density plantations, not one was a product of selfing. According to this, olive would behave as an allogamous species in mono-cultivar growing conditions and the pollen coming from long distances would be able to produce a normal bearing. Therefore, there is strong evidence to support the idea that the cultivars studied could be self-incompatible. Future experiments in self-compatibility should include a paternity check of the possible self seeds obtained.