A method for the enzyme immunoassay (EIA) of delta sleep-inducing peptide (DSIP) has been developed and applied to determine the metabolic clearance rate and biological half-life of DSIP administered to dogs. Antisera to DSIP conjugated to bovine serum albumin were raised in rabbits and proved to be specific for the C-terminus of the peptide. DSIP conjugated to horseradish peroxidase served as the labeled antigen in the EIA and enzyme activity was determined by fluorophotometry. The assay sensitivity was approximately 30 pg/ml. 1 or 2 mg of DSIP was injected intravenously into 4 anesthetized dogs and blood was taken at 5-min intervals. Unextracted plasma was subjected to the EIA directly and showed parallel displacement curves to the standards. DSIP was found to have a rapid disappearance with a mean metabolic clearance rate of 30.7 ± 2.5 ml/kg · min and a mean half-life of 4.0 ± 0.7 min in the dogs. Additional measurements of the metabolic parameters in a monkey and 3 rats treated similarly revealed a rapid in vivo clearance of DSIP from plasma with a half-life of 2.9 and 2.0 ± 0.54 min, respectively.