Primary structure analysis and location of introns suggests evolutionary relatedness among vitellogenin (Vg) genes from vertebrates and invertebrates, including insects. We have determined the complete nucleotide sequence of the gypsy moth VgmRNA, which shows that its structure is significantly different from VgmRNAs in other systems. The nucleotide sequence was determined using overlapping cDNA fragments generated from RACE reactions and rTh polymerase-mediated PCR. The VgmRNA is 5579 nucleotides long and codes for both the large and small subunits. However, the arrangement of the subunit encoding regions in the gypsy moth VgmRNA is opposite of what has been observed in other systems. Gypsy moth Vg gene is the first reported example of a Vg gene where the 5'-terminal region codes for the large subunit and the 3'-terminal region for the small subunit. Also, the sequence near the junction of subunits was significantly different from those found in other insects. This may be responsible for the relatively more stable precursor of Vg subunits found in the gypsy moth hemolymph. It is not clear where this divergence in the structure of Vg gene occurred during evolution, since the Vg gene of another lepidopteran, Bombyx mori, conforms to the structure of those in vertebrates and other invertebrates.