In addition to forming bilayers to separate cellular compartments, lipids participate in vesicular trafficking and signal transduction. Among others, phosphatidic acid (PA) is emerging as an important signaling molecule. The spatiotemporal distribution of cellular PA appears to be tightly regulated by localized synthesis and a rapid metabolism. Although PA has been long proposed as a pleiotropic bioactive lipid, when and where PA is produced in the living cells have only recently been explored using biosensors that specifically bind to PA. The probes that we have generated are composed of the PA-binding domains of either Spo20p or Raf1 directly fused to GFP. In this chapter, we will describe the expression and purification of GST-fusion proteins of these probes, and the use of phospholipid strips to validate the specificity of their interaction with PA. We will then illustrate the use of GFP-tagged probes to visualize the synthesis of PA in the neurosecretory PC12 cells and RAW 267.4 macrophages. Interestingly, the two probes show a differential distribution in these cell types, indicating that they may have different affinities for PA or recognize different pools of PA. In conclusion, the development of a broader choice of probes may be required to adequately follow the complex dynamics of PA in different cell types, in order to determine the cellular distribution of PA and its role in various cellular processes.