In tissue-engineered (TE) heart valves, cell-mediated processes cause tissue compaction during culture and leaflet retraction at time of implantation. We have quantified and correlated stress generation, compaction, retraction, and tissue quality during a prolonged culture period of 8 weeks. Polyglycolic acid /poly-4-hydroxybutyrate strips were seeded with vascular-derived cells and cultured for 4-8 weeks. Compaction in width, generated force, and stress was measured during culture. Retraction in length, generated force, and stress was measured after release of constraints at weeks 4, 6, and 8. Further, the amount of DNA, glycosaminoglycans (GAGs), collagen, and collagen cross-links was assessed. During culture, compaction and force generation increased to, respectively, 63.9% +/- 0.8% and 43.7 +/- 4.3mN at week 4, after which they remained stable. Stress generation reached 27.7 +/- 3.2 kPa at week 4, after which it decreased to similar to 8.5 kPa. At release of constraints, tissue retraction was 44.0% +/- 3.7% at week 4 and decreased to 29.2% +/- 2.8% and 26.1% +/- 2.2% at, respectively, 6 and 8 weeks. Generated force (8-16mN) was lower at week 6 than at weeks 4 and 8. Generated stress decreased from 11.8 +/- 0.9 kPa at week 4 to 1.4 +/- 0.3 and 2.4 +/- 0.4 kPa at, respectively, weeks 6 and 8. The amount of GAGs increased at weeks 6 and 8 compared to week 4 and correlated to the reduced stress and retraction. In summary, prolonged culture resulted in decreased stress generation and retraction, likely as a result of the increased amount of GAGs. These results demonstrate the potential of prolonged tissue culture in developing functional, nonretracting, TE heart valves.