The metastatic murine mammary tumor cell line 410.4 and its nonmetastatic counterpart tumor line 410 were examined for the presence of prostaglandin E2 (PGE2) binding using a 3H-PGE2 ligand binding assay. Inhibition of endogenous prostaglandin synthesis with indomethacin was shown to increase markedly binding of 3H-PGE2. Equilibrium binding data for tumor 410.4 show that specific binding is saturable, reversible by unlabeled PGE2, temperature-dependent and specific. PGE1, PGE2 or 16-16-dimethyl PGE2 compete well with 3H-PGE2 for binding. PGD2 partially inhibits 3H-PGE2 binding, whereas PGA2 does not compete. Scatchard analysis of equilibrium binding data reveals a high affinity (Kd = 3.9 X 10(-9) M) and an average of 33,785 binding sites/cell. In contrast, binding of 3H-PGE2 to nonmetastatic line 410 has a slightly lower affinity (Kd = 8.8 X 10(-9) M) and an average of 368,857 binding sites/cell. 3H-PGE binding to line 410 cells is comparatively nonspecific as PGD2 is nearly as effective as PGE1, PGE2 and an analogue of PGE2 in competing with 3H-PGE2 and PGA2 also inhibits 3H-PGE2 binding.