It was recently reported that human umbilical endothelial vein cells (HUVECs) transdifferentiate and express cardiac genes when co-cultured with rat neonatal cardiomyocytes (Condorelli et al. (2001)). If substantiated and optimized, this phenomenon could have many therapeutic applications. We re-investigated the HUVEC-rat neonatal cardiomyocyte co-culture system but detected cardiomyocyte markers (sarcomeric myosin) in only 1.2% of the cells containing nuclei that were immuno-positive for human nuclear antigen (HNA); and the frequency of such cells was not enhanced in co-cultures containing more embryonic cardiomyocytes. Because the majority of HNA-positive/myosin-positive cells were binucleated, we tested the hypothesis that these cells resulted from HUVEC-cardiomyocyte fusion rather than from HUVEC transdifferentiation. Analysis with a Cre/lox recombination assay indicated that virtually all HUVECs containing cardiac markers had fused with cardiomyocytes. To determine whether human cardiomyocyte genes are activated at low levels in HUVEC-cardiomyocyte co-cultures, quantitative RT-PCR was performed with primers specific for human beta-MyHC and cTnI. We found no evidence for transcriptional activation of these genes. None of our data support conversion of HUVECs to cardiomyocytes.