Various drugs added to the culture medium of rabbit mammary gland were assayed for their capacity to affect the lactogenic and the mammogenic activities of prolactin. Three lysosomotropic agents NH4Cl, chloroquine and methylamine which were previously demonstrated to inhibit the degradation of the hormone-receptor complex after its internalization (down-regulation) did not prevent the initiation of casein synthesis, of lactose synthetase activity and of DNA synthesis. Five microtubule disrupting drugs, colchicine, colcemid, vinblastin, podophyllotoxin and nocodazole inhibited the induction of casein and DNA synthesis by prolactin whereas two inactive analogues, trimethylcolchicinic acid and lumicolchicine had no effect. None of these drugs exhibited any general cytotoxic effect as judged by the capacity of the tissue to incorporate 14C aminoacids into total proteins and 3H-uridine into total RNA. The microtubule disrupting drugs did not greatly reduce the rate of casein synthesis in the cultured mammary tissue explanted from lactating rabbits. The data suggest that the down-regulation of prolactin receptor is not strictly required for the two considered prolactin activities. By contrast, the integrity of microtubules, or at least of structures in which tubulin is involved, is necessary to ensure a normal transmission of the prolactin information responsible for the initiation of milk and DNA synthesis. In addition, the fact that the lactogenic and the mammogenic activities of prolactin are affected by the same drugs suggests that these two properties of the hormone are mediated by cellular mechanisms which have at least one common step.