Fruit production in many fruit tree species is dependent on cross-pollination between varieties. This is caused by the existence of a self-incompatibility mechanism that prevents fertilisation if pollen and style bear the same S-locus specificity. In some species, such as sweet cherry and apricot, self-fertile varieties have been produced by mutagenesis and breeding. The aim of this long-term study was to investigate if the knock-out of one of the pistil S-alleles through genetic engineering results in self-fertility. Plants of the strongly self-incompatible apple cultivar Elstar were transformed with different S-allele knockout constructs. During two subsequent years controlled pollination tests were carried out on the different transgenic lines to determine the strength of their self-incompatibility system. Here we show the results of six transgenic lines bearing an antisense knockout construct. In three lines, the self/cross fruit set ratio was approximately 1, which indicates that the self-incompatibility mechanism in these lines is switched off. Besides these completely self-fertile lines, also a moderately self-fertile line, characterised by intermediate levels of self-fertilisation, was obtained. These results indicate that different levels of self-fertility may be obtained through genetic engineering.