AIM: To use in vivo instead of in vitro cell seeding in heart valve tissue engineering. METHODS AND RESULTS: Intraperitoneally preseeded, photo-oxidized bovine pericardial pulmonary valve constructs (group 1) were compared with non-preseeded constructs (group 2) implanted in sheep. All valves functioned normally and were macroscopically intact at explantation [1 week (n = 6) and 1 month (n = 6) in each group], except for one thrombosed leaflet in a group-2 valve at 1 month. Almost 10-fold higher neomatrix deposition and doubling of the leaflet thickness were found in group 1 vs. 2 (P < 0.05). A concomitant significant decrease in leaflet length (15%) was found at 1 month in group 1. The total cross-sectional surface and total amount of collagen of the original matrix remained unchanged in both groups at all times. Immunohistochemistry showed a low immune response, stem/progenitor cell infiltration, appropriated differentiation, and spontaneous endothelialization of the valves. Significantly, increased re-cellularization was found after IP preseeding compared with spontaneous seeding: cell coverage of the leaflet was 71-100 vs. 8-26% (P < 0.05), respectively. CONCLUSION: Complete re-cellularization can be obtained by IP preseeding of an acellularized cross-linked matrix. Well-functioning valve constructs show cellularization and differentiation into myofibroblast phenotype and concomitant neomatrix deposition.