Background: Embryonic stem cells provide the most promising tool for cell replacement therapy including transplantation of human embryonic stem (hES) cell- derived cardiomyocytes in the infarcted area of the heart. Here we provide data for differentiation of cardiomyocytes from hES cells and firstly describe their hormonal modulation. Methods: Using Micro-Electrode Arrays as a novel electrical mapping technique of beating cardiomyocyte clusters within whole hES cell aggregates, we were able to measure the field potential generation and morphology changes during hormonal modulation. Results: We found that isoproterenol provokes, similar to the mouse ES cell system, a strong positive chronotropic effect with an EC₅₀ of around 10^-8 M. Moreover, isoproterenol stimulated with a higher EC₅₀ value the slow field potential amplitude, FP_slow, indicating a stimulation of Ca²⁺ channels in ventricular-like ES cell-derived cardiomyocytes which is shown to be clearly independent from frequence modulation. In contrast, carbachol (10 µM) produced a transient negative chronotropic effect but had no effect on FP_slow. Conclusion: The Micro-Electrode system allows measurement of ionic channel modulation and chronotropic responsiveness in a pharmacological screening setup. Moreover, all our data indicate that cardiomyocytes derived from human embryonic stem cells exhibit a physiological response to the major hormones of the vegetative nervous system and might therefore serve as an ideal candidate for the use in cell replacement strategies.