Bovine iris and choroid melanosomes at two ages (<1 week and >2 years) wer examined by inductively coupled plasma mass spectrometry (ICP-MS), elemental analysis, infrared spectrometry (IR) and X-ray photoelectron spectrometry (XPS). When iris and choroid melanosomes at the same age were compared, the quantification of metal elements by ICP-MS revealed that choroid melanosomes had a higher binding capacity for the carboxylate-binding metal ions (e.g. Na+, K+, Mg2+, Ca2+ and Zn2+). Elemental analysis showed a higher O:N ratio in choroid melanosomes. Both observations suggested that choroid melanosomes have a higher content of carboxylate-containing monomer than iris melanosomes. IR spectrometric analysis showed a red shift (∼8 cm−1) ofthe absorption peak of aromatic C=C, C=N and C=O at ∼1630 cm−1 in the IR spectrum of iris melanosomes relative to choroid melanosomes. Increased conjugation in the molecular structure of the pigment is proposed to contribute to this peak shift. It is also notable that although the elemental anlaysis showed different C, N and O contents is the two types of melanosomes, XPS showed almost the same elemental compositions on the surface of two types of iris and choroid melanosomes studied. When the melanosomes from the same tissues at different ages were compared, ICP-MS analysis suggested that the number of carboxylate groups in the melanosomes decreased with age. Both elemental analysis and XPS showed that C:N ratio decreased with age, which was proposed to be due to both a decrease in carboxylate groups in mature samples and to the fissure of phenol rings caused by age-associated oxidation. Such age-related oxidative damage diminishes conjugation and is manifested by blue shifts of absorption peaks for aromatic double bonds in the IR spectra of mature melanosomes. XPS analysis showed that the ratio of C-O:C=O decreased with age. These tissue-related and age-related chemical differences between samples affected the optic density and metal binding properties of melanosomes, which are believed to be closely associated with the biological functions of melanins.