The nervous system and digestive tract of workers, soldiers and alate reproductives of Reticulitermes flavipes, the eastern subterranean termite, were examined for neuropeptide F (NPF)-like immunoreactivity using an antibody to Helicoverpa zea midgut peptide I (Hez MP-I). NPF-like material was observed in approximately 70 cells in the brain and over 75 cells of the ventral nerve cord in all castes. Immunoreactive axons originating from the brain and from 15-25 neurosecretory cells on the foregut occurred over the corpora allata (CC)/corpora cardiaca (CA) complex, salivary glands, foregut, and the anterior half of the midgut. Immunoreactive axons on the rectum originated from the terminal abdominal ganglion. Over 600 NPF-like endocrine cells were counted in the midgut of all castes. A radioimmunoassay with the Hez MP-I antibody was used to monitor purification of R. flavipes NPF-like material through 8 HPLC steps from an extract of 117,300 workers (350 g). A partial amino acid sequence was determined by Edman degradation, and PCR was used to amplify a corresponding cDNA with sequence from R. flavipes head and midgut cDNA. The cDNA sequence codes for the following putative translated product: VPSVWAKPSDPEQLADTLKYLEELDRFYSQVARPRFa, which was termed Ref NPF. This sequence possesses several conserved features common to other invertebrate NPFs and even neuropeptide Y-like peptides. This peptide was chemically synthesized and used in juvenile hormone (JH) synthesis and gut motility bioassays. JH bioassays were performed on R. flavipes female brachypterous neotenic corpora allata (CA) incubated in 10 M NPF or Dippu-allatostatin 2 (AST2) 10 M solutions. Ref NPF had no direct effect on JH production by CA, but slightly increased the effect of AST2 on JH inhibition. Ref NPF was also ineffective on the CA of the cockroach Diploptera punctata. The effect of Ref NPF, Ang NPF and Dm NPF were tested on R. flavipes foreguts with an impedance monitor and hindguts with a force transducer. Ref NPF, Ang NPF and Dm NPF did not have an effect on foregut contractions. Hindgut contractions were significantly inhibited with Dm NPF, even when stimulated with serotonin or Leucokinin I. Ref NPF and Ang NPF did not significantly effect hindgut contractions.