Relaxin was localized in the cytoplasm of endometrial gland cells in the uterus of day 60 pregnant guinea pigs with the peroxidase-antiperoxidase method. The immunoprecipitate was located over the apical portions of the glandular cells. Stain was not found over non-endometrial portions of the uterus, endometrial surface epithelium or cells of the uterine cervical glands. Agar double-immunodiffusion analyses demonstrated a reaction of identity when extracts of uterus from day 60 pregnant guinea pigs and a porcine relaxin standard were tested with an antiserum produced against highly purified porcine relaxin. An extract of uteri from day 60 pregnant guinea pigs gave a positive response in the mouse uterus bioassay for relaxin.