Monoclonal antibodies with specificity for the abundant envelope surface glycoprotein (gp67) of Autographa californica nuclear polyhedrosis virus (AcMNPV) were used to screen a lambda gt11 expression library of AcMNPV DNA fragments. The gp67 gene was mapped to the left end of the EcoRI H fragment in a right-to-left orientation on the consensus map of AcMNPV. A 2.1-kilobase transcript which hybridized to the region was first detected in cell extracts at 2 h postinfection; it peaked in abundance at 18 h postinfection and thereafter was present at lower levels. The nucleotide sequence of the region was determined, and a 1,590-nucleotide open reading frame flanked by an AT-rich sequence was identified that could encode a polypeptide with 529 amino acid residues (molecular mass of 60,167 daltons). Computer analysis indicated that the peptide possesses two hydrophobic regions near the N and C termini as well as six potential N-linked glycosylation sites. We suggest that following cleavage of a signal peptide, the polypeptide undergoes further processing and becomes anchored at its C terminus in the virus envelope. The final seven amino acid residues at the C terminus contain basic amino acids and may have a role in virion assembly.