Cholecyst-derived extracellular matrix (CEM) is a novel, proteinaceous biomaterial, derived from the porcine cholecyst, which may have potential applications as a scaffold in the area of heart valve tissue engineering. In this study the potential of CEM to support the proliferation of valvular endothelial cells (VECs) and valvular interstitial cells (VICs), while maintaining their phenotypic mRNA synthesis, protein expression and morphology was assessed by biochemical assays, electron microscopy, immunostaining and reverse-transcriptase polymerase chain reaction. VICs and VECs were isolated from the porcine aortic valve and techniques were developed for the isolation of CEM for cell culture. VECs and VICs cultured on CEM adhered and proliferated, maintaining their phenotypic morphology. VECs synthesised von Willebrand factor mRNA and endothelial nitric oxide synthase (eNOS) mRNA and expressed eNOS and VICs synthesised alpha-smooth muscle actin (alphaSMA) mRNA and expressed alphaSMA. Cellular area fraction of VICs expressing alphaSMA was 87.7+/-6.8% and cellular area fraction of VECs expressing eNOS was 93.8+/-9.3%. Findings of this study support the hypothesis that CEM is a potential biomaterial for tissue engineered heart valve scaffold design.