The mouse M-twist gene codes for a basic helix-loop-helix protein which was shown to be inhibitory for differentiation of myogenic cells in culture. Mouse embryonic stem (ES) cells of line BLC6 efficiently differentiating into skeletal muscle cells when cultivated as embryo-like aggregates (embryoid bodies) were stably transfected with the plasmid pME18s-twist containing the M-twist gene under the control of the modified SV40 early promoter SR alpha. Two pME18s-twist-expressing clones showed delayed and reduced skeletal muscle cell differentiation depending on the level of exogenous M-twist expression compared to control cells. By morphological analysis using phase contrast microscopy and hematoxylin-eosin staining, the development of first myocytes and formation of myotubes in embryoid body outgrowths of these clones were found to be delayed for about 3 days in comparison to control cells. Immunofluorescence studies with a monoclonal antibody against sarcomeric myosin heavy chain revealed that myogenic cells appeared in so-called myogenic centers showing a reduced number of myocytes and myotubes in the M-twist-expressing clones. Using RT-PCR analysis the expression of the skeletal muscle determination genes myf5, myogenin, and MyoD as well as muscle-specific genes coding for the gamma-subunit of the nicotinic acetylcholine receptor and the cell adhesion molecule M-cadherin were found to appear with a delay of at least 1 to 4 days in the pME18s-twist-transfected cells during the development of embryoid bodies. We conclude that the constitutive expression of the mouse M-twist gene during ES-cell-derived differentiation has an inhibitory effect on skeletal muscle cell development depending on the level of exogenous M-twist expression.