Alkaline phosphatase (AP) activity is an accepted marker of epithelial cell differentiation and integrity in intestinal mucosal explant systems. In experiments involving AP it is usually expressed as activity per unit protein in tissue culture. It has been previously demonstrated that during organ culture a considerable amount of protein and nucleic acid (NA) may leave the cell and enter the surrounding medium. It may therefore not be appropriate to measure enzyme synthesis in relation to total tissue protein. In this study we attempted to gather additional information on enzyme dynamics and tissue integrity during organ culture of intestinal mucosal ex-plants from children with celiac disease (CD) on a normal diet (ND) (n = 21), children with CD on a gluten-free diet (GFD) (n = 12), and patients with gastrointestinal disorders other than CD (controls) (n = 17). We confirmed the leakage of protein, NA, and AP from the tissue explant into the medium, and normalized the AP data by expressing AP as total AP in tissue plus medium per total NA in tissue plus medium. In patients with CD on a ND, the AP level at time 0 was 9.58 ( ± 12.2) mIU/μg NA and after 24 h culture rose to 21.9 ( ± 15.7) mIU/μg NA. In patients with CD on a GFD the baseline AP level was 24.9 ( ± 34.8) mIU/μg NA, and this value rose after culture to 28.6 ( ± 17.9) mIU/μg NA. In the controls the initial AP activity was 17.2 ( ± 15.7) mIU/μg NA, and the increase was to 38 ( ± 27.6) mIU/μg NA. When we related tissue AP levels alone to tissue NA levels, the activity after 24 h incubation was lower than in the preincubation sample. Our study indicates that AP activity in organ culture should be expressed as total AP per total NA.