Eye formation is an important feature for various cheese varieties. This study firstly aimed to evaluate the potential of X-ray computed tomography (CT) and image analysis software as a non-invasive method to quantify cheese eye volume. The quantification of the eye volume by CT was validated with 12 eyeless hard cheeses made with the inclusion of 0-100 hollow PP balls (Ø = 10 or 20 mm). The results obtained for the total volume of the 'artificial eyes' showed a good correlation with the volume of the added balls (R 2 > 0.998). In a second part of the study, the developed CT method was applied for the non-invasive investigation of the eye formation in semi-hard Tilsit-type cheese with adjuncts of Lactobacillus casei or Lactobacillus plantarum. Both adjuncts completely metabolized citrate in cheese, thus enhancing gas production. Total eye number, relative eye volume and the size distribution of eyes were determined in order to reveal the influence of the two adjunct cultures on eye formation. In comparison to the control, the addition of L. casei resulted in a significantly higher eye number and eye volume. In contrast, the addition of L. plantarum did not influence the eye formation in a significant way, though it yielded cheeses with more succinate and aspartate and less serine at significant levels. The results imply that, apart from CO 2 production, other factors, such as the presence of eye nuclei and the dynamics of CO2 diffusion, interfere in an important way in the process of eye formation in cheese.