This study was undertaken to reveal the quantitative distribution of the insect neuropeptide periviscerokinin-1 (Pea-PVK-1) in the central nervous system of Periplaneta americana and to demonstrate that neurons stained in a previous immunohistochemical study contain authentic Pea-PVK-1. For this, we combined ELISA, HPLC, and MALDI-TOF mass spectrometry. The high specificity of the used antiserum enabled the quantification of Pea-PVK-1 in unseparated tissue extracts. No cross-reactivities with other insect neuropeptides were detected in ELISA. Only two immunoreactive fractions, coeluting with synthetic Pea-PVK-1 in its oxidized and nonoxidized form, were found in HPLC-separated extracts of the brain, suboesophageal ganglion, metathoracic ganglion, second abdominal ganglion with or without perisympathetic organ, and terminal ganglion. By using MALDI-TOF mass spectrometry, we were able to confirm the existence of authentic Pea-PVK-1 in these fractions. The abdominal perisympathetic organs contained 6.3 pmol Pea-PVK-1 per animal; another 1.3 pmol were found in the abdominal ganglia. More than 90% of the total 8.2 pmol in the central nervous system was found in the abdominal ganglia and their perisympathetic organs. The corpora cardiaca and corpora allata did not contain immunoreactive material, suggesting that Pea-PVK-1 is not released by the cephalic neurohaemal system. The quantitative distribution of Pea-PVK-1 differs considerably from that of other known insect neuropeptides.