The phospholipids present in uveal (iris/ciliary body and choroid) and retinal bovine ocular melanosomes were identified using mass spectrometry. Similar phospholipid content is found for the two types of uveal melanosome, with sphingomyelin being the major species. Significant differences are found between the uveal and retinal melanosome. Glycerophosphoethanolamine (GPEtn) is the major species in the retinal pigment epithelium (RPE); 93% of the GPEtn contain polyunsaturated fatty acids, notably docosahexanoic acid and arachidonic acid, in the sn-2 position. RPE melanosomes also contain detectable quantities of glycerophosphoserine and glycerophosphate; these species were not detected in the uveal samples. While the structural and functional roles of melanosomal lipids largely remain to be determined, these different lipid compositions reported herein offer new insights into the roles of melanosomes in the different ocular tissues.