Pluripotent embryonic stem (ES) cells spontaneously differentiate via embryo-like aggregates into cardiomyocytes of pacemaker-, atrium- and ventricle-like type, which can be distinguished by their specific patterns of action potentials. It has been shown that retinoic acid (RA) treatment during ES cell differentiation increases the number of cardiomyocytes in a time- and concentration-dependent manner. In order to test the effect of RA on cardiomyocyte differentiation and specialization into ventricle-like cardiomyocytes, we studied gene expression of β-galactosidase driven by the ventricular myosin light chain-2 (MLC-2v) promoter as an indicator for ventricular differentiation. Clones containing the stably integrated expression vector pGNA/MLC-2.1 were selected, which revealed an increase of β-galactosidase activity in cardiomyocytes of embryoid bodies at day 7 + 16. RA, both, in the all-trans and in the 9-cis configuration resulted in a significant acceleration of cardiomyocyte differentiation and a transient increase of β-galactosidase activity. To test whether this acceleration of cardiac differentiation and RA-induced increase of the MLC-2v promotor/β-galactosidase activity reflects an increase of cardiac- and ventricle-specific gene expression, a semi-quantitative RT-PCR analysis was performed for α-cardiac myosin heavy chain (α-MHC) and MLC-2v genes. It was shown that both 10^-8 M and 10^-9 M RA resulted in an increased level of α-cardiac MHC and MLC-2v mRNA in embryoid bodies in early, but not in terminal developmental stages. This led us to the conclusion that the RA-induced accelerated expression of cardiac-specific genes results in an enhanced development of ventricular cardiomyocytes. An increased number of ventricle-like cells after RA treatment was also found by patch-clamp analysis. The number of cardiomyocytes with Purkinje- and ventricle-like properties was shown to be increased by RA, whereas the number of pacemaker- and atrium-like cells was reduced and early pacemaker cells were not quantitatively affected.