Vascular smooth muscle cell (VSMC) differentiation is important in understanding vascular disease; however, no in vitro model is available. Totipotent mouse embryonic stem (ES) cells were used to establish such a model. To test whether the ES cell‐derived smooth muscle cells expressed VSMC‐specific properties, the differentiated cells were characterized by 1) morphological analysis, 2) gene expression, 3) immunostaining for VSMC‐specific proteins, 4) expression of characteristic VSMC ion channels, and 5) formation of [Ca²⁺]_i transients in response to VSMC‐specific agonists. Treatment of embryonic stem cell‐derived embryoid bodies with retinoic acid and dibutyryl‐cyclic adenosine monophosphate (db‐cAMP) induced differentiation of spontaneously contracting cell clusters in 67% of embryoid bodies compared with 10% of untreated controls. The highest differentiation rate was observed when retinoic acid and db‐cAMP were applied to the embryoid bodies between days 7 and 11 in combination with frequent changes of culture medium. Other protocols with retinoic acid and db‐cAMP, as well as single or combined treatment with VEGF, ECGF, bFGF, aFGF, fibronectin, matrigel, or hypoxia did not influence the differentiation rate. Single‐cell RT‐PCR and sequencing of the PCR products identified myosin heavy chain (MHC) splice variants distinguishing between gut and VSMC isoforms. RT‐PCR with VSMC‐specific MHC primers and immunostaining confirmed the presence of VSMC transcripts and MHC protein. Furthermore, VSMC expressing MHC had typical ion channels and responded to specific agonists with an increased [Ca²⁺]_i. Here we present a retinoic acid + db‐cAMP‐inducible embryonic stem cell model of in vitro vasculogenesis. ES cell‐derived cells expressing VSMC‐specific MHC and functional VSMC properties may be a suitable system to study mechanisms of VSMC differentiation.—Drab, M., Haller, H., Bychkov, R., Erdmann, B., Lindschau, C., Haase, H., Morano, I., Luft, F. C., Wobus, A. M. From totipotent embryonic stem cells to spontaneously contracting smooth muscle cells: a retinoic acid and db‐cAMP in vitro differentiation model. FASEB J. 11, 905–915 (1997)