Pituitary glands of beef, sheep and pig origin were successively extracted with water at pH 5.5, M/10 (NH4)2SO4 at pH 4.0, M/4 (NH4)2SO4 at cither pH 5.5 or 7.5, and finally with 60 percent ethanol at pH 9.8 to 10. The extracts were bioassayed for the anterior pituitary hormones and for the proteinases I and II. With certain exceptions, the first extract contained mainly FSH and proteinase I; the second, LH and TSH, the third, GH and proteinase II; and the fourth, prolactin. FSH and GH were further purified to establish the amenability of these extracts to fractionation. Growth hormone isolated from beef pituitary extracts prepared at pH 5.5 appears to be a mixture of two biologically active proteins: one with alanyl and phenylalanyl NH2-terminal groups and another with methionyl and alanyl NH2-terminal groups. Small amounts of NH2-terminal serine and glutamic acid were also present. By varying the conditions of extraction appropriately, the amounts of the two types of hormones could be varied inversely. On prolonged extraction at pH 5.5 and 20°, the growth hormone with NH2-terminal methionine had almost completely replaced that with NH2-terminal phenylalanine. It is concluded that methionyl growth hormone is an artifact of the isolation procedure and is derived from the phenylalanyl hormone by the hydrolytic action of previously undescribed proteinases, optimally active at pH 5 to 6.5. On the basis of sedimentation, NH2-terminal, and methionine analyses, the methionyl growth hormone does not differ significantly in molecular weight from conventional growth hormone. Furthermore, it is inferred that of the seven bonds in the hormone containing methionine only the one nearest the NH2-terminus in the phenylalanyl chain is cleaved. Two new proteolytic enzymes were found in the pituitary. One hydrolyzes the Ser-Met- bond in Ser-Met-Glu (NH2) and CH3CO-Ser-Tyr-Ser-Met-Glu(NH2) and has a hydrolytic optimum at pH 4.5 to 5. The other readily hydrolyzes the -Met-Glu (NH2) bond in the tripeptide but has relatively little action on the pentapeptide. Its optimum is at pH 6 to 7.0. Sheep extracts yield growth hormone having the usual phenylalanl and alanyl NH2-terminal groups. Since sheep extracts obtained at pH 5.5 also contain proteinases capable of hydrolyzing methionyl peptides, the absence of a methionyl NH2-terminal group in the isolated sheep growth hormone is interpreted to mean that peptide bonds susceptible to the proteinases are absent or inaccessible. Bovine growth hormone isolated from M/4 (NH4)2SO4 extracts prepared at pH 5.5 was characterized by a higher electrophoretic mobility than that isolated from alkaline extracts. Treatment with mildly alkaline solutions produced a partial conversion to a slower electrophoretie component whose mobility approximated that of growth hormone prepared from alkaline extracts.