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A convenient method was established for the separation of the inner layer (lamina perivitellina) and the outer layer (lamina extravitellina) of the vitelline membrane. The method involved incubation of the egg yolk in .01-/V HC1 for 1 h at 37 C, which allowed the two layers to separate intact.Electron microscopic inspection showed that the separated layers retained the structural integrity of the original membrane and that the "continuous membrane" (lamina continud) was attached to the outer layer. Chemical analysis showed that the outer layer contained twice as much carbohydrate as the inner layer. After solubilization of the separated layers in 1 % sodium dodecyl sulfate (SDS), composition of macromolecular components was determined.The inner layer consisted of three major soluble glycoproteins (GP)-32% GP-I, 45% GP-II, and 20% GP-III-in addition to the SDS-insoluble glycoprotein (3%), designated as GP-IV. The outer layer contained 43% ovomucin, 37% lysozyme, and 20% vitelline membrane outer I (VMOI). The inner and outer layers were found to contribute almost equally to the physical strength of the whole vitelline membrane. When the whole membrane and the inner and outer layers were treated with . \-M mercaptoethanol for 1 h (37 C, pH 7.0), membrane strengths decreased by 77%, 51%, and 78%, respectively. This indicated that the physical strength of the whole membrane is primarily maintained by disulfide-bonded components in the outer layer. Because removal of lysozyme and VMOI, the major soluble components of the outer layer, did not affect membrane strength, it was concluded that disulfide bonds of ovomucin were essential to structural integrity of the vitelline membrane. (

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