A cDNA clone representing the gene for the E3 glycoprotein, the haemagglutinin, of bovine coronavirus was isolated from a plasmid cDNA library of the viral genome and sequenced. The gene is located immediately 5' of the E2 glycoprotein gene on the viral genome. Nucleotide sequencing of the E3 gene predicts a polypeptide of 424 amino acids with an Mr of 47K. In vitro translation of mRNA transcribed from the cloned E3 gene yielded a polypeptide of Mr 45K, similar to that predicted from the nucleotide sequence. In the presence of microsomal membranes, the in vitro product was cotranslationally processed to a 62K polypeptide which comigrated on SDS-polyacrylamide gels with the E3 monomer (gp62) obtained from virus-infected cells. Both the 45K and 62K polypeptides were immunoprecipitated with E3-specific monoclonal antibodies, confirming the identity of the gene as that encoding the E3 glycoprotein. Finally, only monoclonal antibodies to the E3 protein inhibited haemagglutination by the virus thus confirming its identity as the haemagglutinin of bovine coronavirus.