Neuropeptide Y-like immunoreactivity (NPY-LI) in dog plasma was characterized and quantified using three extraction methods (Sep-Pak:acetonitrile, HCl:ethanol, and ethanol). Sep-Pak extraction yields the best recovery and preserves the integrity of the peptide. Oxidized NPY is not generated during blood collection. Using two antisera of different specificities, at least three peptide forms in normal dog arterial and venous plasma were detected. A peptide with retention times similar to oxidized NPY or peptide YY is the major component of plasma NPY-LI under basal conditions, but NPY(1-36) predominates during sympathetic stimulation. The mature peptide in dog plasma is similar to human NPY. The antiserum ABII provides a more accurate measure of circulating NPY(1-36) and its oxidized form. The antiserum ABI is useful for detecting NPY-like fragments.