The immune system protects an organism in a multitude of different ways. The T lymphocytes, with their respective T Cell Receptor (TCR), bear a grave role by binding foreign antigens and initiating the adaptive immune response. Even though the TCR and its associated proteins, CD3 and zeta, are long known to execute this vital task, much remains to be elucidated. The chain represents an essential part of the complex for assembly and membrane association, as well as for accurate signal initiation. Most studies on the TCR/CD3/zeta complex were made by rather indirect methods, such as co-immunoprecipitation. However, with the recent progress made in light microscopy, enabling single molecule observations, more direct experiments can be done. In this work, single molecule tracking experiments were performed to get information on the diffusional behaviour of some of the involved proteins, i.e. TCRbeta and zeta. Furthermore, two-color PALM was used to examine possible clustering of TCR beta and zeta in resting and stimulated primary mouse T cells. The utilized algorithms for localization and tracking were evaluated before use and found to be adequate for densities and SNRs in the range of the actual experiments. The potency of the used murine T cells to induce Ca2+ flux by pMHC binding was confi rmed. Interestingly, the diffusional behaviour of zeta was found to differ from the one of TCRbeta , highlighting the possibility of TCR/CD3-free zeta within the membrane. These diffusional differences between zeta and TCRbeta indicate less stable interactions within the TCR/CD3/zeta complex, than identi fied by biochemical methods. Furthermore, hints on spatial clustering of both proteins were identifi ed. However, crucial pit-falls within a commonly used data analysis on PALM images, i.e. Ripley's K function, were shown.