A method developed for separation and partial purification of human pituitary hormones has been applied to acetone-dried horse pituitary glands. Growth hormone, follicle-stimulating hormone and luteinizing hormone activities were each obtained in separate fractions. Further purification of each hormone was achieved by the same or slightly modified procedures effective for the corresponding human hormones and the yields and potencies of the preparations were comparable to those obtained by other investigators. Hormones prepared from acetone-dried horse pituitaries stored for 25 years were of similar yield and potency to those prepared from recently collected glands. Horse and human luteinizing hormones cross-reacted immunologically and the horse hormone was assayed by an immunological method. Strong cross-reactions were observed between horse, pig and beef growth hormones and a very weak cross-reaction between horse and human growth hormones.