Former work has shown that crude extracts of ovine pineal glands probably exert a stimulating activity on the release of gonadotropins of anterior pituitaries in vitro. By aqueous extraction followed by ultrafiltration through anisotropic membranes high Mr (above 100,000 daltons) fractions were obtained, which exhibit a stimulating effect on the levels of gonadotropins in the medium of either cultured pituitary cells or anterior hemipituitaries in short-term culture. Partial purification of a pineal luteinizing hormone release stimulating factor was accomplished by Sephadex G-150 filtration with a biopotency of 226 +/- 23 micrograms LH-RP-1 equivalents per mg protein and without an affinity for binding to anti-LHRH or anti-LH antibodies. The present data substantiate that high Mr forms, slightly heavier than authentic pituitary LH (Mr 23,000 daltons) and therefore not identical to the hypothalamic decapeptide LH-RH, represent ovine pineal factors which can increase the concentration of LH in the medium of cultured anterior pituitaries, but does not influence the secretion of prolactin in vitro.