Open reading frame (ORF) 3 of the genome of lactate dehydrogenase-elevating virus (LDV), strain P, was cloned into the plasmid pcDNAI/Amp and in vitro transcribed and translated. Translation of ORF 3 yielded a soluble protein of the expected size (about 21 kDa). When synthesized in the presence of endoplasmic reticulum (ER) membranes the resulting glycoprotein of about 36 kDa became associated with the membranes. However, disruption of the ER vesicles by incubation in carbonate buffer, pH 11.5, resulted in the release of the protein from the membranes. Hydrophobic moment analysis of the ORF 3 protein indicated the absence of any potential transmembrane segments, except for a N-terminal signal peptide, but no cleavage of the signal peptide was observed during membrane-associated in vitro synthesis. The ORF 3 protein elicited a strong antibody response in infected mice. The antibodies from infected mice as well as a monoclonal antibody specifically precipitated the in vitro-synthesized ORF 3 protein, but no protein from LDV virions. The overall results suggest that the ORF 3 protein is a nonstructural, highly glycosylated, and antigenic glycoprotein that is probably soluble and secreted or at most only weakly associated with membranes via the signal peptide.