Changes in prolactin (PRL)-like bioactivity in rat milk after a longer isolation of litters and the effects of oxytocin (OT) were determined by Nb2 lymphoma cell bioassay and compared with the immunoreactivity using an enzyme immunoassay. First, we confirmed the appropriate dilution rates of rat milk which could neutralize the influence of an antimitogenic factor(s) in milk to Nb2 lymphoma cell proliferation. To prolong the litter removal, i.e. 6 h to 20 h, resulted in the increase in an antimitogenic factor(s) for Nb2 lymphoma cells in milk and in the significant decrease in milk PRL concentration measured by both assays, and plasma bioactive PRL level increased. The bioactivity/immunoreactivity ratio of PRL in milk accumulated in the mammary gland was 0.65 in rats after a 20-h isolation, but 1.18 after a 6-h isolation. The milk PRL concentration in rats isolated for 6 h decreased significantly within 45 min after the administration of OT and the plasma PRL concentration increased only slightly, but OT had no effect in rats after isolation for 20 h. The present findings indicate that milk PRL might transfer to plasma with the excessive engorgement of milk in the mammary gland and with OT administration. Moreover, PRL trapped in milk for a long period loses its biological activity more rapidly than its immunological activity, since bioactivity/immunoreactivity ratio in the 20-h isolation group was significantly lower than in the 6-h isolation group.