Both PtdIns(3,4,5)P3 (PIP3) and atypical protein kinase C (aPKC) play central roles in the polarization of many cell types. In epithelial cells, both PIP3 and aPKC are required for the development of apico-basolateral membrane polarity. However, the relationship between PIP3 and aPKC during the establishment and maintenance of polarized membrane domains remains to be clarified. We show that depolarized MDCK cells retain a polarized basal distribution of PIP3, supporting a role for PIP3 in determining the basolateral membrane domain. Importantly, overexpression of a kinase-negative mutant of aPKC lambda (aPKC lambda kn) impaired the basal distribution of PIP3, indicating that aPKC kinase activity is required for the restriction of PIP3 to the basal region. In support of this, overexpression of aPKC lambda kn during polarization, but not after polarization, caused whole membrane distribution of PIP3 as well as defects in epithelial polarization. (C) 2008 Elsevier Inc. All rights reserved.