Testosterone (T) has a strong anabolic effect on skeletal muscle and is believed to exert its local effects via the androgen receptor (AR). The AR harbors a polymorphic stretch of glutamine repeats demonstrated to inversely affect receptor transcriptional activity in prostate and kidney cells. However, longer AR glutamine repeat lengths are associated with greater lean body mass and higher serum T in humans. The effects of AR glutamine repeat length on skeletal muscle are unknown. Purpose:To determine the effects of AR glutamine repeat length on AR function in skeletal muscle cells. Methods:AR expression vectors carrying 14, 24, and 33 glutamine repeats, respectively, were constructed and AR transcriptional activity was determined in transfected C2C12 myoblasts using an AR sensor plasmid. Each vector was subsequently stably transfected into C2C12 cells to create 3 independent cell lines: C2C12AR14, C2C12AR24, and C2C12AR33. Cellular proliferation and creatine kinase (CK) activity were determined. Gene expression was assessed via RT-PCR. Myosin expression, myotube formation, and myonuclear fusion index were examined immunohistochemically. Results: Transcriptional activity increased with increasing repeat length (3.91±0.26 vs. 25.21±1.72 vs. 36.08±3.22 relative light units in AR14, AR24, and AR33, respectively; p<0.001), in response to T. Ligand activation ratio indicated significant ligand-independent AR transcriptional activity. Significant AR protein expression was only detected in AR14 myoblasts. In contrast, AR mRNA expression was elevated in each stable line in the myoblast stage and throughout differentiation. The proliferation of AR33 cells was significantly decreased vs. AR14 (20512.3±1024.0 vs. 27604.17±1425.3, p<0.001) after 3 days. The CK activity of AR14 cells was decreased in comparison to AR24 and AR33 cells (54.9±2.9 vs. 68.3±2.2 and 70.8±8.1 units/μg protein, respectively; p<0.05) after 5 days of differentiation. The myonuclear fusion index was lower for both AR14 (15.21±3.24%, p<0.001) and AR33 (9.97±3.14%, p<0.001) in comparison to WT C2C12 cells (35.07±5.60%). Both AR14 and AR33 cells displayed atypical myotube morphology. RT-PCR revealed differences in the expression of genes involved in differentiation, cell fusion, and cell cycle progression. Conclusion: AR polyglutamine repeat length affects receptor activity and alters the growth and development of C2C12 cells. This polymorphism may explain some of the heritability of muscle mass in humans.