AbstractNeurochemical differences in the hypothalamic‐pituitary axis between individuals and between ages may contribute to differential susceptibility to cocaine abuse. This study measured peptide levels in the pituitary gland (Pit) and lateral hypothalamus (LH) in adolescent (age 30 days) and adult (age 65 days) mice from four standard inbred strains,FVB/NJ,DBA/2J, C57BL/6J, andBALB/cByJ, which have previously been characterized for acute locomotor responses to cocaine. Individual peptide profiles were analyzed using mass spectrometric profiling and principal component analysis. Sequences of assigned peptides were verified by tandem mass spectrometry. Principal component analysis classified all strains according to their distinct peptide profiles in Pit samples from adolescent mice, but not adults. Select pro‐opiomelanocortin‐derived peptides were significantly higher in adolescentBALB/cByJandDBA/2J mice than inFVB/NJor C57BL/6J mice. A subset of peptides in theLH, but not in the Pit, was altered by cocaine in adolescents. A 15 mg/kg dose of cocaine induced greater peptide alterations than a 30 mg/kg dose, particularly inFVB/NJanimals, with larger differences in adolescents than adults. Neuropeptides in theLHaffected by acute cocaine administration included pro‐opiomelanocortin‐, myelin basic protein‐, and glutamate transporter‐derived peptides. The observed peptide differences could contribute to differential behavioral sensitivity to cocaine among strains and ages.imagePeptides were measured using mass spectrometry (MALDI‐TOF) in individual lateral hypothalamus and pituitary samples from four strains and two ages of inbred mice in response to acute cocaine administration. Principal component analyses (PCA) classified the strains according to their peptide profiles from adolescent mice, and a subset of peptides in the lateral hypothalamus was altered by cocaine in adolescents.