The purpose of this Study was to study the effect of calcium, cyclic AMP (cAMP) and cyclic GMP (cGMP) on embryonic stem cell (ESC) motility during TNF-alpha-induced chemotaxis. ESCs were monitored using a chemotaxis chamber, with different concentrations of calcium or cAMP or cGMP added to the medium. Changes in intracellular calcium ([Ca2+](i)) were measured with the fluorescent dye fura-2/AM. We combined migratory parameters in a mathematical model and described it as 'mobility". After adding Calcium, a dose-dependant increase in cell speed was found. Cyclic AMP increased mobility as well as the [Ca2+](i). In contrast, adding dbcGMP resulted in a significant decrease in the mobility of the ESCs. During migration ESCs showed an increase in [Ca2+](i). Furthermore, TNF-alpha dramatically increased the movement as well as the directionality of ESCs. These results demonstrate that ESCs are highly motile and respond to different concentrations of calcium in a dose-related manner. (C) 2009 Elsevier Inc. All rights reserved.