β₁ integrin-deficient (β₁^-/-) ES cells showed increased differentiation of cardiac cells characterized by reduced adhesion and high beating frequency. Whereas in whole embryoid body outgrowths of β₁^-/- cells maximum levels of α_v, β₃ and β₅ integrin mRNA were delayed and transiently upregulated, in cardiac clusters isolated from β₁^-/- cells, only β₃ integrin mRNA levels were enhanced in comparison to wild-type (wt) cells. To answer the question, whether α_v and β₃ integrins may compensate, at least partially, the loss of β₁ integrin function during cardiac differentiation, the distribution of α_v and β₃ integrins in β₁^-/- and wt pacemaker-like cardiac cells was analyzed. A different distribution of α_v and β₃ integrins in β₁^-/- v wt cardiac cells was found. In wt cardiac cells. β₁ integrin was localized in specialized subsarcolemmal regions, in particular, at focal contacts and costameres, but α_v integrin was diffusely distributed. In contrast, in β₁^-/- cardiac cells, α_v integrin was preponderantly localized at cell membranes, focal contacts and costameres. β₃ integrin displayed a diffuse pattern both in wt and in β₁^-/- pacemaker-like cells at early differentiation stages, whereas at terminal stages, β₃ was colocalized with sarcomeres in wt. but not in β₁^-/- pacemaker-like cells. Quantitative immunofluorescence analysis revealed increased α_v and β₃ integrin levels in β₁^-/- pacemaker-like cardiac cells. Our results led us to conclude that altered cellular distribution of α_v integrin and upregulation of β₃ integrin correlate with growth and survival of β₁^-/- cardiac pacemaker-like cells at an early developmental state. However, α_v and β₃ integrins cannot functionally compensate the loss of β₁ integrin during terminal differentiation of cardiac cells implicating that cardiomyocytes require specific β₁ integrin functions for cardiac specialization.