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Transforming growth factor beta 1 (TGF-β1) is a key morphogen in regenerative endodontics; yet, its location within the hard tissue phase of dentin and its availability in mature roots have not been fully elucidated.Young mature (n = 8) and immature (n = 11) roots from sound premolars were obtained from 13 orthodontic patients aged 17 ± 1 and 12 ± 1 years, respectively. Roots were cleaned of organic remnants in 5% sodium hypochlorite. The width of the minor foramen was measured using a digital microscope. TGF-β1 distribution was assessed in 3 roots per group by immunostaining combined with confocal laser scanning microscopy. The root dentin of the remaining 13 roots was powdered and decalcified in 17% EDTA to determine the overall levels of hard tissue–embedded TGF-β1 by enzyme-linked immunosorbent assay. Data were compared between groups using the Student t test (α = .05).The minor foramen was 168 ± 49 μm versus 557 ± 295 μm in mature compared with immature roots (P < .05). TGF-β1 was highly stainable toward the pulp space in both groups. It was clearly associated with peritubular dentin and apparently absent in nontubular outer dentin. TGF-β1 content was 115 ± 31 pg and 74 ± 35 pg/100 mg mature versus immature root dentin, respectively (P > .05).TGF-β1 is deposited into the peritubular dentin. It should be possible to release this molecule in regenerative endodontic procedures from young mature roots as well as immature roots.