Successful cellular cardiomyoplasty is dependent on biocompatible materials that can retain the cells in the myocardium in order to promote host tissue repair following myocardial infarction. A variety of methods have been explored for incorporating a cell-seeded matrix into the heart, the most popular options being direct application of an injectable system or surgical implantation of a patch. Fibrin-based gels are suitable for either of these approaches, as they are biocompatible and have mechanical properties that can be tailored by adjusting the initial fibrinogen concentration. We have previously demonstrated that conjugating amine-reactive homo-bifunctional polyethylene glycol (PEG) to the fibrinogen prior to crosslinking with thrombin can increase stability both in vivo and in vitro. Similarly, when mesenchymal stem cells are combined with PEGylated fibrin and injected into the myocardium, cell retention can be significantly increased and scar tissue reduced following myocardial infarction. We hypothesized that this gel system could similarly promote cardiomyocyte viability and function in vitro, and that optimizing the mechanical properties of the hydrogel would enhance contractility. In this study, we cultured HL-1 cardiomyocytes either on top of plated PEGylated fibrin (2D) or embedded in 3D gels and evaluated cardiomyocyte function by assessing the expression of cardiomyocyte specific markers, sarcomeric -actin, and connexin 43, as well as contractile activity. We observed that the culture method can drastically affect the functional phenotype of HL-1 cardiomyocytes, and we present data suggesting the potential use of PEGylated fibrin gel layers to prepare a sheet-like construct for myocardial regeneration. Biotechnol. Bioeng. 2015;112: 1446-1456. (c) 2015 Wiley Periodicals, Inc.