Endothelial cell coverage of blood-contacting devices is crucial to their eventual success in the clinic. Two established human cell lines derived from HUVEC (human umbilical vascular endothelial cells), CRL 2922 and CRL 2873, have been widely utilized to study and model endothelial cell biology. However, it is not clear if these two cell lines would be useful for modeling primary endothelial cell interaction with newly-formulated biomaterials in tissue engineering applications. Hence, this study was conducted to compare the adhesion and proliferation characteristics of HUVEC grown on seven different substrata, tissue culture polystyrene (TCPS), gelatin, chitosan, poly-L-lysine, hyaluronan, poly-L-lactic acid (PLLA), and polylactic-co-glycolic acid (PLGA). The short-term adhesive behavior (2 h) of HUVEC on the various substrata was not closely-replicated by either CRL 2873 or CRL 2922. This was likely because the 2 h time-frame is too short for identification of differences in the interaction among the three cell types grown on various substrata. There was much faster proliferation of CRL 2922 on all seven substrata when compared to HUVEC and CRL 2873. Moreover, the proliferation rates of CRL 2922 on the various substrata showed little variation. In contrast, HUVEC and CRL 2873 displayed similar trends in proliferation rates, with gelatin and TCPS yielding the highest rates, and PLLA and PLGA yielding the lowest rates. Hence, CRL 2873 is better suited for modeling primary endothelial cell interaction with newly-formulated biomaterials than CRL 2922. The advantage of using CRL 2873 over HUVEC for biomaterial screening is that it is immortalized and displays much less inter-batch variability than primary culture.