We describe the use of molecules labeled with short-lived emitting radionuclides for molecular imaging in combination with the positron emission tomography technique. How to use molecular probes to visualize and quantitatively determine rates of specific biochemical events such as synaptic transmission, enzymatic processes and binding to specific receptor proteins is highlighted. The sensitivity of the PET technique and the ability to measure and validate relationships between molecular events and biological functions is a key factor for the successful application of PET in biomedical research. In specific applications, the opportunity of using molecules labeled in specific positions may be critical. Molecular imaging using PET is also gaining increasing interest as a tool in drug development, especially when applied to early proof of concept studies in man. In this chapter, the concept of molecular imaging is exemplified and the use of position-specific labeling of tracer molecules as a tool to gain understanding of complex biological processes will be discussed.