Defects in meiotic spindle structure can lead to chromosome segregation errors and genomic instability. In this study the potential role of protein kinase C delta (PKCdelta) on meiotic spindle organization was evaluated in mouse oocytes. PKCdelta was previously shown to be phosphorylated during meiotic maturation and concentrate on the meiotic spindle during metaphases I and II. Currently we show that when phosphorylated on Threonine 505 (pPKCdelta(Thr505)), within the activation loop of its C4 domain, PKCdelta expression was restricted to the meiotic spindle poles and a few specific cytoplasmic foci. In addition, pPKCdelta(Thr505) co-localized with two key microtubule organizing center (MTOC)-associated proteins, pericentrin and gamma-tubulin. An interaction between pPKCdelta(Thr505) and pericentrin as well as gamma-tubulin was confirmed by co-immunoprecipitation analysis using both fetal fibroblast cells and oocytes. Notably, targeted knockdown of PKCdelta expression in oocytes using short interfering RNAs effectively reduced pPKCdelta(Thr505) protein expression at MTOCs and leads to a significant (P < 0.05) disruption of meiotic spindle organization and chromosome alignment during MI and MII. Moreover, both gamma-tubulin and pericentrin expression at MTOCs were decreased in pPKCdelta(Thr505)-depleted oocytes. In sum, these results indicate that pPKCdelta(Thr505) interacts with MTOC-associated proteins and plays a role in meiotic spindle organization in mammalian oocytes.