A method has been developed for separation and partial purification of four protein hormones from acetone-dried human pituitary glands. The major portion of growth hormone, follicle-stimulating hormone, luteinizing hormone and thyroid-stimulating hormone activities are each obtained in a separate fraction. Although the yields of the individual hormones are similar to those obtained by other investigators, the present procedure is simpler and a cleaner separation of the hormones has been achieved. Methods for further purification of each fraction are indicated including preparation of luteinizing hormone of very high potency (equivalent to 5.3mg. of standard NIH-LH-S3/mg.).