LY309887 is significantly cytotoxic against the human leukemia cell line CCRF-CEM (IC50: 9.9 nM). LY309887 also has high affinity at human folate receptor (FR)α and FRβ (Ki, 1.78 nM, and 18.2 nM, respectively) .

In mice bearing C3H mammary cancer cells, LY309887 (3 mg/kg-100 mg/kg, i.p.) displays complete inhibition on the tumor growth.