OBJECTIVE: To quantify component volumes (mineral, organic, total water, firmly and loosely bound water volumes, and permeability) at histological points in bovine enamel and investigate their association with bovine enamel's optical behavior and fracture toughness (FT). DESIGN: In vitro, cross-sectional, and experimental study. Longitudinal ground sections of ten permanent bovine incisors were analyzed using microradiography, polarizing microscopy, microhardness tester, and confocal laser fluorescence microscopy (CLFM). Continuous data were analyzed using regression and correlation tests. RESULTS: Bovine enamel exhibited gradients in both composition and FT from the surface inward, with the inner third showing positive birefringence in water immersion and opaque areas under immersion in air (explained by composition gradients), along with intermediate FT. Bovine enamel presented mineral volume of 79.6 % ( ± 2.7 %), water volume of 11.4 % ( ± 20.4 %), organic volume of 9.1 % ( ± 2.4 %), and permeability of 6.5 % ( ± 0.5 %). Water loss upon air drying was 1/5th of that in human enamel, representing a novel finding in enamel biology. The organic gradient was confirmed by CLFM. The ratio of firmly bound water to organic content was negatively associated with fracture toughness across the enamel layer. CONCLUSION: bovine enamel differs from human enamel in terms of composition, birefringence, and permeability, requiring caution when interpreting results from studies using it to test dental products intended for humans. The composition of bovine enamel fully explained its optical behavior and partially explained its FT gradient.