Immature skeletal muscle cells, or myoblasts, have been used in cellular cardiomyoplasty, which attempts to regenerate cardiac muscle tissue by injecting cells into damaged myocardium. In some studies, muscle tissue within myoblast implant sites may be morphologically similar to cardiac muscle. We hypothesized that identifiable aspects of the cardiac milieu may contribute to the growth and development of implanted myoblasts in vivo. To test this hypothesis, we designed a novel in vitro system to mimic some aspects of the electrical and biochemical environment of native myocardium. This system enabled the separation of the 3-dimensional, electrical and biochemical signals that may be involved in myoblast proliferation and plasticity. Myoblast cells were grown on 3-D polyglycolic acid mesh scaffolds under control conditions, in the presence of cardiac-like electrical current fluxes, or in the presence of culture medium that had been conditioned by mature cardiomyocytes. Cardiac-like electrical current fluxes caused increased myoblast number in 3-D culture, as determined by DNA assay. The increase in cell number was due to increased cellular proliferation and was not due to differences in apoptosis, as determined by PCNA and TUNEL staining. Cardiomyocyte-conditioned medium also caused significant increases in myoblast proliferation. Expression of transcription factors governing differentiation along either skeletal or cardiac lineages were evaluated by immunoblotting. Though these assays are qualitative, no changes in differentiation state along either skeletal or cardiac lineages were observed in response to electrical current fluxes. Furthermore, from these experiments, conditioned medium did not appear to alter the differentiation state of skeletal myoblasts. Hence, cardiac milieu appears to stimulate proliferation, but does not affect differentiation of skeletal myoblasts.