To clarify the subspecies-specific functions of protein kinase C (PKC), we constructed cDNAs encoding gamma-, epsilon- and delta-PKC fused with green fluorescent protein (GFP). All fusion proteins had enzymological and immunological characteristics similar to those of native PKCs. When expressed in CHO-K1 cells, each fusion protein showed a specific subcellular localization. Their translocations induced by various stimulation were also diverse. For example, ATP translocated gamma-, epsilon- and delta-PKC-GFP in the cytoplasm to the plasma membrane within 30 sec with a return to the cytoplasm in 3 min, whereas TPA induced slow and irreversible translocation of all subspecies to the plasma membrane. Fatty acids also induced the translocation of gamma- and epsilon-PKC-GFP, but the two PKC subspecies showed distinct translocation and sensitivity to various fatty acids. Furthermore, we revealed that the PKC translocation requires neither the kinase activity of PKC nor its association with cytoskeletal proteins such as F-actin. These results indicate that each subspecies has a spatially and temporally different targeting mechanism that depends on the extracellular and intracellular signals, contributing to the subspecies-specific functions of PKC. These remarkable findings also indicate that a system for monitoring the PKC translocation is a powerful tool for investigating the subspecies-specific functions of PKCs and mechanism of its translocation.