Seventy-five percent of the myoblasts transplanted in the mouse muscle die during the first 4 days following transplantation. The purpose of this study was to determine if anoikis plays a role in this phenomenon. Survival and proliferation of myoblasts in vitro were determined by Hoescht-PI labeling and cell counts respectively. In vivo cell survival and proliferation were quantified by injecting human male myoblasts labeled with 14C-thymidine in SCID mouse muscles. Survival and proliferation of the transplanted myoblasts were evaluated by scintigraphy and quantitative PCR of human Y chromosomal DNA. Inclusion of the extracellular matrix protein fibronectin enhanced transplanted myoblast survival by 1.7-fold while vitronectin improved their proliferation by 1.8-fold. Reductions in FADD and Bit1 expression reduced anoikis in vitro and improved the injected myoblast survival in vivo. Ectopic expression of the anti-apoptotic protein Bcl-2 completely abolished myoblast anoikis in vitro and enhanced cell survival by 3.1-fold in vivo. Cell death following transplantation appears to me mediated in part by anoikis. Inclusion of extracellular matrix proteins enhanced both survival and proliferation. Reduced expression of the proapoptotic proteins Bit1 and FADD or overexpression of Bcl-2 improved myoblast survival.