The effects of adenovirus type 5 infection on the differentiation of cultured human skeletal myoblasts and of myoblast differentiation on the replication of adenovirus were investigated. Although infection of myoblasts concurrently with differentiation induction was inhibitory, myoblast differentiation was not impeded when infection was carried out 2 hr or later after induction. Similar studies conducted with EIA mutant viruses (d1312, pm975, and d11500) revealed that complete inhibition was dependent on the product of 13 S El A transcript expression, although partial inhibition could be induced by the 12 S product. Differentiation of myoblasts results in the generation of multinucleated myotubes and quiescent mononuclear cells. The three cell types (myoblasts, myotubes, mononuclear cells) were differentially permissive to adenovirus infection. The precursor myoblasts and the multinucleated myotubes were found to be permissive for adenovirus infection. The kinetics of their infection was delayed 24-48 hr relative to that of HeLa cells. Quiescent mononuclear cells in the differentiated myoblast cultures were found to be inefficient in supporting the production of adenovirus particles, despite the accumulation of adenovirus DNA and capsid proteins. Host protein synthesis in the three cell types also responded differently to adenovirus infection. In the multinucleated myotubes, host protein synthesis was potently inhibited by adenovirus at late times after infection, whereas it persisted in the proliferating myoblasts and quiescent mononuclear cells.