Androgen signaling is critical for normal fetal development, but is not thought to regulate events in early embryogenesis. Given the interest in factors controlling the differentiation of embryonic stem cells, we have explored the possibility that androgens may play a role. This study demonstrates expression of androgen receptor (AR) RNA and protein in four independent mouse embryonic stem (mES) cell lines and demonstrates that the AR is functional and can interact with transfected androgen response elements (ARE) to promote green fluorescent protein (GFP) expression. AR mRNA was detected throughout 10 days of differentiation in embryoid bodies (EBs). Exposure of EBs to testosterone (T) or dihydrotestosterone (DHT), at doses of 1µM and 0.1µM respectively, promoted formation of beating cardiomyocytes. Flow cytometric analyses demonstrated a significant increase in the number of -actinin and tropomyosin (cardiac markers) positive cells following these treatments. Addition of flutamide (1µM) to T-treated EBs inhibited the T-induced proliferation of cardiomyocytes, confirming that, in this instance, androgens act via the classical AR-mediated genomic pathway. We also report that ES cells express key steroidogenic enzymes, as detected by RT-PCR, and during 24 hour incubations secrete T at concentrations of 1.38 ± 0.22nM, levels comparable to those secreted by cultured Leydig cells. These novel data demonstrate the capacity of androgens to stimulate increased differentiation of mES cells to cardiomyocytes, and is in keeping with recent observations that AR-deficient mice exhibit cardiac impairment in adulthood.