The role of protein kinase C (PKC) isoforms in the commitment of multipotent fibroblasts to the adipocyte lineage and in their terminal differentiation into mature adipocytes was investigated. Ectopic overexpression of PKC-ε, but not other PKC isoforms, committed multipotent NIH-3T3 cells to adipogenic differentiation in the presence of hormonal inducers. In committed 3T3-F442A preadipocytes, PKC-ε protein expression increased during the course of terminal differentiation and cell-permeable PKC-ε inhibitory peptides, which prevent interaction with RACK (receptor for activated C-kinase) proteins, severely inhibited differentiation. PKC-ε accumulated in the nuclei of 3T3-F442A cells shortly after induction of differentiation and exhibited a distinctive punctate speckling immunocytochemical staining pattern. The spatiotemporal aspects of PKC-ε localization and expression coincided with that of C/EBP-β, a transcription factor critically involved in promoting the early phase of adipogenesis. Collectively, these results demonstrate a role for PKC-ε in both adipogenic commitment and preadipocyte terminal differentiation.