Using the polymerase chain reaction (PCR) a 0.8-kb product was amplified from cDNA made from the midgut and Malpighian tubules of fifth instar larvae of Helicoverpa virescens. This 0.8-kb PCR product was then used to isolate a clone of the B subunit of the V-type ATPase from a cDNA library made from the same tissues. The cDNA clone encodes for a protein of 55 kDa which shows very high amino acid homology to other known B subunits of V-type ATPases. The transcript size of the B subunit in the midgut of H. virescens was 2.3 kb, and a transcript of identical size was also detected in the Malpighian tubules. Northern blot analysis revealed the presence of a homologous transcript of 2.6 kb in the midgut of Manduca sexta and PCR analysis also confirmed the presence of such a transcript in the Malpighian tubules and the nervous system of M. sexta, and in the midgut Malpighian tubules of Culex quinquefasciatus. The presence of the V-type ATPase in the Malpighian tubules of lepidopteran insects suggests that the transport of ions across the cell membrane in this tissue is also probably driven by a similar process as that observed in the midgut of these insects.