Throughout life, cell migration plays an important role in wound healing, tissue regeneration and cancer growth. CTL (cytotoxic T lymphocytes) are an important component of the immune system responsible for the elimination of virus-infected or tumorigenic cells. In order to reach and kill their target cells, CTL have to migrate actively within the damaged tissue. We used time-lapse microscopy to study the migration of primary human CTL in absence of target cells in vitro at 37 °C. We analyzed the two-dimensional trajectories of CTL on fibronectin substrate and at varying external calcium concentrations. We tracked the position of migrating CTL during 1 h and determined mean displacement, instantaneous speed and duration and frequency of moving vs. resting phases. Track analysis revealed that CTL migrate randomly with an average velocity of 8.8 mm/min. The cells do not migrate continuously, but often interrupt their movement, change their shape and become round before moving again. A reduction of the free external calcium concentration by addition of 1.2 mM of the calcium buffer EGTA to the cell culture medium did not affect cell viability but caused a decrease of the average velocity to 6.6 mm/min and increased the duration and frequency of the resting phases. These findings indicate that CTL may be well adapted to operate in tissues with low external calcium concentration which could be helpful to fight against cancer and infection.