We have studied the membrane topography and N-glycosylation of the envelope proteins of lactate dehydrogenase-elevating virus (LDV, strain P). Transcripts of open reading frames (ORFs) 2, 5, and 6 were in vitro translated in the absence and presence of microsomal membranes, and the products analyzed for molecular weight, sensitivity to endoglycosidase F/N-glycosidase F and proteinases, and reaction with anti-LDV antibodies. The ORF 6 mRNA translation was enhanced in the presence of microsomal membranes. ORF 6 encodes a polytopic class III membrane protein identified as the nonglycosylated virion envelope protein (M/VP-2; approximately 18 kDa). The protein has a very short (about 11 amino acids) ectodomain, a longer (about 79 amino acids) C-terminal endodomain, and crosses the membrane three times between these domains. ORF 5 encodes the primary virion envelope glycoprotein (VP-3P) (25-42 kDa). Our results suggest that it is a polytopic class I glycoprotein. After removal of a signal peptide, the processed protein of about 171 amino acids consists of a short (approximately 30 amino acids) N-terminal ectodomain with three asparagine residues that appear to be N-glycosylated, a segment that crosses the membrane three times, and an about 74 amino acid long C-terminal endodomain. Neutralizing anti-LDV antibodies are probably directed to an epitope(s) in the N-terminal ectodomain. The ORF 2 protein is a standard class I glycoprotein with a single C-terminal membrane anchor segment and its signal peptide is removed during membrane-associated synthesis. The remaining ectodomain (about 165 amino acids) contains three asparagine residues which appear to be N-glycosylated. Our results suggest that the ORF 2 protein may be present in a low concentration in LDV virions (VP-3M).