Anabolic androgenic steroids (AAS) lead to cardiac complications, and relationship among AAS, tumor necrosis factor alpha (TNF-α), and renin-angiotensin system (RAS) has been reported. Apoptosis is implicated in the shifting of the hypertrophic heart to the heart failure. The testosterone (T) in anabolic concentration induces apoptosis in cardiac myocytes. However, little is known about the relationship among testosterone, TNF-α, and RAS in this process. We investigated whether the apoptosis and the caspase-3 activity induced by testosterone in anabolic concentration involves the participation of the angiotensin converting enzyme (ACE) and TNF-α in cardiac myocyte cell culture (H9c2, ATCC). Cardiomyocytes were treated with testosterone (100 µM), doxorubicin (DOX; 5 µg/mL), T + etanercept (Eta; 100 µg/mL), T+losartan (Los; 10-7 M), and T+ Ac-DEVD-CHO (200 µM). The treatment time with testosterone alone or in combination in culture wells was 72 h. Cell viability was evaluated by the MTT assay in different concentrations of T (0,5-200 µM). Apoptosis was determined by flow cytometry, proteolytic activity of caspase-3, TNF-α quantification and ACE activity by ELISA method. Values are expressed as the mean ± S.E.M, compared by ANOVA, followed by the post-hoc Tukey test. T elicit a concentration-dependent decrease in cell viability (0.5 µM: 12.49±1.54%, 5 µM: 21.36±0.94%, 50 µM: 30.06±1.38%, 100 µM: 64.74±1.16%, 200 µM: 74.85±1.97%), and co-treatments with Eta (5.3±1.7%), Los (6.4±1.8%) or Ac-DEVD-CHO (12.9±3.5), reduced the effect of T in anabolic concentration (100 µM; 64.7±1.2%). T also induces apoptosis (concentration-dependent)(Figure 1) and increase in the proteolytic activity of caspase-3, which were reduced by the co-treatments (Figure 2). TNF-α and ACE activity were elevated by T treatment, and co-treatment with Los and Eta were able to reduce these effects (Figure 2). It is concluded that there is an interaction between testosterone, Ang II and TNF-α in inducting apoptosis and caspase-3 activity in cultured cardiac myocytes, which contributes to reducing the viability of these cells (cytotoxic effect) induced by testosterone in anabolic concentration.