Objectives Collagen barrier membranes are used in guided bone regeneration/guided tissue regeneration because of their excellent bio‐ and cytocompatibility. However, they are considered to have limitations in clinical outcome because of rapid and unpredictable degradation profiles. The aim of this study was to investigate the degradation behavior of two porcine‐based, non‐cross‐linked collagen membranes in vitro and in vivo. Materials and Methods Remaix™ (RX; Matricel GmbH, Herzogenrath, Germany) and Bio‐Gide® (BG; Geistlich Pharma AG, Wolhusen, Switzerland) membranes were characterized by testing mechanical strength, denaturation temperature, enzymatic degradation and hydroxyproline content in vitro (n = 5 up to 16). Thereafter, both membranes were implanted subcutaneously in rats (n = 20) for up to 20 weeks to investigate tissue compatibility with respect to membrane thickness. Results BG contained a significant higher hydroxyproline content compared with RX, but RX showed a higher stress at break (dry: 11.4 (SD 2.9) vs. 5.5 (SD 1.5) N/mm²), higher suture retention (wet: 5.6 (SD 1.3) vs. 2.7 (SD 0.7) N), increased denaturation temperature (55.1 (SD 1) vs. 49.4 (SD 0.6)°C) and an almost twofold reduction in degradation rate (15.6% (SEM 1.3)/h vs. 24.8% (SEM 2.9)/h) in vitro. In the rat model, both membranes showed excellent tissue compatibility without signs of inflammatory reactions. Shortly after implantation, RX and BG showed moderate infiltration of mononuclear cells that appeared not to be influenced by the surface texture of the membranes. In the histomorphometric analysis, both membranes showed significant different thickness over the 20 weeks period (P = 0.0002). Although the thickness remained almost stable during the first 9 weeks after implantation, after 20 weeks, the thickness of RX decreased only slightly, whereas BG showed a thickness loss of around 50% and stronger degradation than RX. Therefore, the higher stability of RX against biodegradation found in vitro was confirmed in the animal study. Conclusion This study shows differences in the biodegradation characteristics of two non‐cross‐linked collagen membranes in vitro and in vivo. Whether the higher stability of RX is of clinical relevance should be analyzed in future clinical investigations.