Phosphatidic acid (PA) is one of the major phospholipids in the plasma membrane. Although it has been reported that PA plays key roles in cell survival and morphology, it remains unknown when and where PA is produced in the living cell. Based on the principle of Förster resonance energy transfer (FRET), we generated PA biosensor, and named Pii (phosphatidic acid indicator). In these biosensors, the lipid-binding domain of DOCK2 is sandwiched with the cyan fluorescent protein and yellow fluorescent protein and is tagged with the plasma membrane-targeting sequence of K-Ras. The addition of synthetic PA, or the activation of phospholipase D or diacylglycerol kinase at the plasma membrane, changed the level of FRET in Pii-expressing cells, demonstrating the response of Pii to PA. The biosensor also detected divergent PA content among various cell lines as well as within one cell line. Interestingly, the growth factor-induced increment in PA content correlated negatively with the basal PA content before stimulation, suggesting the presence of an upper threshold in the PA concentration at the plasma membrane. The biosensor also revealed uneven PA distribution within the cell, i.e. the basal level and growth factor-induced accumulation of PA was higher at the cell-free edges than at the cell-cell contact region. An insufficient increase in PA may account for ineffective Ras activation at areas of cell-cell contact. In conclusion, the PA biosensor Pii is a versatile tool for examining heterogeneity in the content and distribution of PA in single cells as well as among different cells.