Integrin cell surface receptors play an important role for cell adhesion, migration, and differentiation during embryonic development by mediating cell-cell and cell-matrix interactions. Less is known about the function of integrins during commitment and lineage determination of early embryogenesis. Homozygous inactivation of the β₁ integrin gene results in embryonal death in mice around the time of implantation. In vitro, differentiation of embryonic stem (ES) cells which lack β₁ integrin (β₁(- /-) into the cardiogenic lineage is delayed and results in a disordered cellular specification (Fassler et al., J. Cell Sci. 109, 9989-2999, 1996). To analyze β₁ integrin function during myogenesis and neurogenesis we studied differentiation of β₁(-/-) ES cells via embryoid bodies into skeletal muscle and neuronal cells in vitro. β₁(-/-) cells showed delayed and reduced myogenic differentiation compared to wildtype and heterozygous (β₁(+/-)) ES cells. RT-PCR analysis demonstrated delayed expression of skeletal muscle-specific genes in the absence of β₁ integrin. Immunofluorescence studies with antibodies against the sarcomeric proteins myosin heavy chain, titin, nebulin, and slow C-protein showed that myotubes formed, but their number was reduced and the assembly of sarcomeric structures was retarded. In contrast, neuronal cells differentiating from β₁(-/-) ES cells appeared earlier than wildtype and heterozygous (β₁(+/- )) ES cells. This was shown by the accelerated expression of neuron-specific genes and an increased number of neuronal cells in β₁(-/-) embryoid bodies. However, neuronal outgrowth was retarded in the absence of β₁ integrin. No functional difference between wildtype and β₁(-/-) cells was found with respect to secretion of γ-aminobutyric acid, the main neurotransmitter of ES cell-derived neuronal cells. The lineage-specific effects of loss of β₁ integrin function, that is the inhibition of mesodermal and acceleration of neuroectodermal differentiation, were supported by differential expression of genes encoding lineage-specific transcription factors (Brachyury, Pax-6, Mash1) and signaling molecules (BMP-4 and Wnt-1). Because of the reduced and delayed expression of the BMP-4 encoding gene in β₁(-/-) cells, we analyzed in wildtype and β₁(-/-) cells the regulatory role of exogenously added BMP- 4 on the expression of the mesodermal and neuronal marker genes, Brachyury and wnt-1, respectively. The data suggest that BMP-4 plays a regulatory role during differentiation of wildtype and β₁(-/-) cells by modifying mesodermal and neuronal pathways. The reduced expression of BMP-4 in β₁(- /-) cells may account for the accelerated neuronal differentiation in β₁(- /-) ES cells.