Bovine placental lactogen (bPL), a polypeptide hormone functionally related to bovine growth hormone (bGH) and bovine prolactin (bPL), has been isolated from placentas by pH and ammonium sulfate precipitation, gel filtration, and ion exchange chromatography on DEAE- and CM-cellulose. The hormone has been purified to approximately 99% homogeneity, as determined by end group analysis. On disc gel electrophoresis at pH 9.0 bPL migrates as a pair of closely spaced bands (Rf = 09517 and 0.541) between the positions of bGH and bPR. Its molecular weight, as estimated by gel filtration on Sephadex G-200 in 6 M guanidine hydrochloride and 6.5 mM dithiothreitol, is 22, 150 and its isoelectric point is 5.9. The amino acid composition of bPL closely resembles that of bGH and bPR except for a higher content of serine and glycine and a lower leucine content. Like bPR, it has 2 tryptophans and 6 cysteines, but its COOH-terminal sequence is identical with that of bGH: -Cys-Ala-Phe-OH. By Ouchterlony immunodiffusion, bPL forms lines of partial identity with bGH against bGH antisera and with ovine placental lactogen (oPL) against oPL antisera. In the bPL-antibPL system, oPL forms a line of partial identity while bGH and bPR do not cross-react. However, bPL does not form a precipitin line with bPR antisera. These data would indicate that in terms of structure, and hence molecular evolution, bPL and other subprimate placental lactogens occupy a position more intermediate between growth hormone and prolactin than do the primate placental lactogens.