The reuse of electrophysiology catheters could result in significant cost savings. However, re-sterilization of catheters could lead to several adverse consequences, including toxicity related to potential chemical reactions that occur during sterilization due to toxic residues remaining on the surface of catheters. The objective of this study was to investigate in vitro cytotoxicity of polyurethane (PU)-based catheter extracts on macrophages after their re-sterilization. We have compared three sterilization methods: steam autoclave, ethylene oxide (EtO) and hydrogen peroxide plasma (Sterrad system). Our results showed that the viability of cells varied from 90% to 99% as a function of incubation time and number of sterilization cycle. While there was no statistical difference based on the sterilization procedure, the number of sterilization cycles (up to 10 cycles) presents a statistically significant effect on the viability of J774 macrophages. However, extract obtained after resterilization of PU-based catheters had a low cytotoxic effect on J774 macrophages, since the overall cell mortality remained under 10%. An inhibitory effect on cell growth was also observed, which was not significant either as a function of incubation time, sterilization technique or the number of sterilization cycles. Finally, extracts of PU-based catheters had no statistically significant effect on TNF-alpha release by J774 macrophage. Even though there were some statistically significant differences between the control and processed samples, and among processed samples, our data suggest that one single reprocessing of PU-based catheters may not induce clinically significant changes in their cytotoxicity behaviour.