Cellular interactions with substrata of the microenvironment are one of the major mechanisms for differentiation and morphogenesis. Many of these interactions are mediated via the β₁ integrin subfamily of cell surface receptors, which are believed to transduce signals upon cell adhesion. We have used β₁ integrin-deficient embryonic stem cells to test their ability to differentiate into cardiac muscle cells. We show here by several approaches that β₁ integrin is important for normal cardiogenesis. First, the in vitro differentiation of β₁ integrin-deficient embryonic stem cells into cardiac muscle cells is retarded. This is demonstrated by the delayed expression of cardiac muscle-specific genes and action potentials. Second, the specification of cardiac precursor cells into pacemaker-, atrial- and ventricular-like cells is significantly impaired in β₁ integrin-deficient cells. The occurrence of atrial- and ventricular-like cells is reduced and transient. Only cells exhibiting pacemaker-like action potentials of high frequency and arrhythmias survive. Third, the sarcomeric architecture is incomplete and disarranged in the absence of β₁ integrin. Fourth, β₁-deficient embryonic stem cells can contribute to the developing heart in chimaeric mice but many areas with β₁-null cells contain cell debris. The number of β₁-null cells decreases from prenatal to postnatal stages and is lost completely in 6-month-old hearts. Thus, we conclude that interactions with the extracellular matrix via β₁ integrin is necessary for differentiation and the maintenance of a specialized phenotype of cardiac muscle cells.