Despite systemic prophylaxis, infection rates after orthopedic surgery can reach more than 1%. A new HAP/TCP bone substitute loaded with 125 mg of gentamicin was designed for prophylactic use. Its aim was to enhance the efficacy of systemic prophylactic treatments by increasing the local antibiotic concentration. For prophylactic applications, release had to take place within 48 hours not to select antibiotic-resistant bacterial strains. The purpose of this study was to investigate the releasing mechanisms of gentamicin from the porous HAP/TCP matrix. The release rate of gentamicin trough the porosities of the bone substitute was investigated in vitro, in 0.9% sodium chloride solution. The rate appeared to be related to the bone substitute volume and fit classical diffusion laws. All the gentamicin was released in less than 48 hours: this rate corresponds to the recommendations for the prophylactic use of antibiotics.