Background. Among the various complications of heart transplantation (HTx), the vasculopathy of the allograft (CAV), a phenomenon of chronic rejection, is still a serious problem. Recently, the literature has shown that low testosterone levels in men are associated with cardiovascular disease. In this study, we evaluated the influence of testosterone plasma levels on CAV development. Methods. We studied, with a prospective observational study, all consecutive male HTx patients evaluated from May 2010 to June 2011 at our center. All subjects underwent accurate medical history collection, physical examination, biochemical blood tests, hormone levels, transthoracic Doppler echocardiography, coronary flow velocity reserve assessment, and coronary angiogram. Results. HTx subjects with CAV had significant lower total testosterone plasma levels (12.9 +/- 3.9 vs. 15.8 +/- 5.8 nmol/L), free testosterone (0.26 +/- 0.07 vs. 0.31 +/- 0.08 nmol/L), and coronary flow velocity reserve (2.35 +/- 0.60 vs. 2.81 +/- 0.78 s) with respect to No-CAV patients. Considering the patients as a whole group, a significant negative relation was found between free and total testosterone plasma levels and some cardiovascular risk factors (cholesterol and fasting blood glucose). A significant linear inverse relation was found between total and free testosterone plasma levels and CAV grading. Only free testosterone plasma levels were independent predictors for CAV. Conclusions. We showed for the first time the influence of testosterone plasma levels on CAV development: indirectly increasing traditional risk factors and directly with a probable influence on alloimmune response.