Neuropeptide FF (NPFF) is a peptide with opioid modulating and cardioexcitatory effects, it is present in the central nervous system and in the periphery of several mammalian species. Using a sensitive and specific radioimmunoassay for NPFF-like immunoreactivity (NPFFir) we observed that the peptide concentration fluctuated in a pattern compatible with pulsatile secretion of the peptide in human blood. When NPFF samples were collected every 2 or 5 min for a 95 min period in healthy volunteers the basal NPFF concentration in human blood was 2.2 +/- 0.5 pg/ml and the NPFF pulses (14.6 +/- 10.6 pg/ml) represented a 526 +/- 280% increase over baseline. The NPFF pulses where short, suggesting a rapid degradation of NPFF in the circulation. We observed no twenty-four hour rhythm of NPFF in human blood when NPFF samples were taken during one day every four hours. Fluctuations in NPFF levels found in the 95 min and the 24 h studies did not correlate with plasma vasopressin levels. Our study did not support the concept that vasopressin and NPFF may be co-released from the pituitary. However, the pulsatile character of NPFF secretion in itself suggests a biologic role for neuropeptide FF in humans.