Five neuropeptides with C-terminal amino acid sequence homology to cockroach allatostatins have been identified in the blowfly Calliphora vomitoria. Three have the same pentapeptide C-terminal amino acid sequence as allatostatin 1 of the cockroach Diploptera punctata. A hexadecapeptide designated callatostatin 1, isolated from thoracic ganglia, brains, and heads, has the sequence Asp-Pro-Leu-Asn-Glu-Glu-Arg-Arg-Ala-Asn-Arg-Tyr-Gly-Phe-Gly-Leu-NH2. Callatostatins 2 and 3 have been isolated from heads and thoracic ganglia, respectively; they comprise the last 14 and 8 residues of callatostatin 1. Callatostatin 4, isolated from thoracic ganglia, has the sequence Xaa-Arg-Pro-Tyr-Ser-Phe-Gly-Leu-NH2, where Xaa is either Asp or Asn. This peptide, with a serine substitution for glycine at position 5, has a C-terminal pentapeptide sequence identical to that of allatostatins 3 and 4 of D. punctata. Callatostatin 5, with the sequence Gly-Pro-Pro-Tyr-Asp-Phe-Gly-Met-NH2, was identified from whole flies. All five peptides inhibit juvenile hormone production by the corpora allata of D. punctata in vitro. Callatostatin 5 was the most potent allatostatin so far tested in this species, with maximum inhibition occurring at 1 nM. In contrast, none of the callatostatins or the allatostatins showed allatostatic activity in mature female C. vomitoria when tested at concentrations of 100 to 0.1 microM. In accordance with these results, immunoreactivity to an antiserum directed against the common C terminus of callatostatin 1 and allatostatin 1 was observed in the corpora allata of D. punctata but not in the corpus allatum of C. vomitoria, despite its presence in neurons of the brain. Neurons in the thoracic ganglion of C. vomitoria that are immunoreactive against this antiserum project to the hindgut, rectum, rectal papillae, and oviduct, suggestive of a function different from that of a true allatostatin.