To test the hypothesis that enamel biomineralization is regulated by sequential expression of an intrinsic genetic program, we designed experiments to determine if a serumless, chemically-defined medium is permissive for position-dependent ameloblast differentiation and subsequent enamel tissue-specific biomineralization in vitro. In the absence of serum or other exogenous growth factors, Swiss Webster strain mouse embryonic (15- and 16-days gestation) mandibular first molar tooth organs (cap stage) developed within 21 days in vitro into well-defined molar tooth organs expressing dentine and enamel biomineralization. Analysis of data obtained from von Kossa histochemistry for calcium salt formation, as well as ultrastructural information obtained from x-ray microanalysis, electron diffraction, transmission electron microscopy and scanning electron microscopy documented tissue-specific patterns of calcium hydroxyapatite formation in the absence of serum within organotypic cultures in vitro. An as yet unknown intrinsic genetic program regulates enamel formation in vitro.