The major protein (PO) in PNS myelin is an integral membrane glycoprotein with a molecular weight of about 30 K. The level of PO protein in the developing sciatic nerve of the chicken was monitored by a solid-phase immunoassay and densitometry of Coomassie blue stained polyacrylamide gels. The most rapid rate of accumulation of PO protein occurred after 16 days of embryonic development. In addition to the 30 K PO protein, a number of higher molecular weight proteins could be distinctly detected by immunoblotting. Amongst these high molecular weight proteins, a species with an apparent molecular weight of 42 K was specifically immunostained with epitope-selected polyclonal antibodies against PO protein. This 42 K protein could be first detected after 16 days of embryonic development and increased rapidly following the pattern of myelination in the sciatic nerve. The enzyme endoglycosidase F, which specifically removes N-asparagine linked high mannose and complex carbohydrates from glycoproteins, converted the PO and 42 K proteins to lower molecular weight forms, which could be specifically immunostained by epitope selected polyclonal antibodies to the PO protein. Subcellular fractionation of the 17-day embryonic nerve demonstrated that the 42 K protein was enriched in myelin and microsomal subfractions relative to the total homogenate. These results indicate that the 42 K immuno-crossreactive protein might be chemically and functionally related to the PO protein of the PNS myelin.