The in vitro effect of wortmannin, an inhibitor of PI3 kinase, on prolactin (PRL) stimulated P70S6K, iodide transport, and milk product synthesis were investigated in cultured mouse mammary tissues. Mouse mammary gland explants were initially incubated for 24 hr in media M199 containing 1 μg/ml insulin and 10−7M cortisol. A subsequent treatment with wortmannin impeded, in a dose-dependent fashion, the PRL stimulation of casein, lipid, and lactose synthesis as well as the PRL stimulation of iodide transport. Rapamycin (25 ng/ml), an inhibitor of P70S6K, also inhibited the effect of PRL on iodide transport; this drug was earlier shown to inhibit PRL effects on milk product synthesis. These results suggest the possible involvement of P70S6K and PI3-kinase in PRL-stimulated milk product formation and iodide transport in mouse mammary explants. Since wortmannin caused a diminished cellular content of P70S6K and a reduced extent of P70S6K migration in polyacrylamide gels (likely due to dephosphorylation), PI3-kinase likely lies upstream in the PRL signaling pathway for P70S6K activation.