The localization of the cyclin-dependent kinase inhibitor p27(kip1) is dependent on the phosphorylation of one of three key amino acid residues: S10, T157 and T198. However, it was unclear whether endogenous p27(kip1) is phosphorylated at T198 in the living cell. In the present work we describe the generation and characterization of a polyclonal antibody able to recognize recombinant, transfected as well as endogenous T198-phosphorylated p27(kip1). Using this antibody, we demonstrate that: (1) endogenous p27(kip1) is phosphorylated at T198 in 4 breast cancer cells lines (MCF7, MDA-MB231, MDA- MB436 and MDA-MB468); (2) T198 phosphorylation is increased in breast cancer cells compared with normal mammary epithelial cells (HMEC); (3) T198-phosphorylated p27(kip1) is exclusively cytoplasmic; (4) T198 phosphorylation is dependent on the activity of the PI3K-PKB/Akt pathway, being it drastically reduced by the pharmacological PI3K inhibitor LY294002 or stimulated by the constitutive activation of PKB/Akt. Finally, in primary human breast carcinomas, cytoplasmic accumulation of T198-phosphorylated p27(kip1) parallels Akt activation. We conclude that in breast cancer cells p27(kip1) is phosphorylated at T198 in a PI3K/Akt dependent manner and that this phosphorylation may contribute to p27(kip1) cytoplasmic mislocalization observed in breast cancer.