The goal of this study was to establish and validate a protocol for preparing bovine cardiomyocytes from slaughterhouse material for nuclear transfer experiments. The cardiomyocyte was selected because it is a terminally differentiated cell and strongly expresses a unique subset of genes which can be monitored during the reprogramming period. A total of 39 trials were conducted, and an optimized protocol was developed yielding individual contractile cardiomyocytes from 3–5-month-old bovine fetuses The basic protocol involves stabilization of bovine heart tissue for transportation from the slaughterhouse to the laboratory by perfusion with Custodiol^®. This was followed by an enzymatic dissociation with collagenase in calcium-free medium and yielded individual contractile rod-shaped cardiomyocytes. Subsequent addition of Ca²⁺ caused the cardiomyocytes to round up which was an essential pre-condition for drawing them into glass transfer pipettes for delivery into the perivitelline space and for efficient electrofusion with cytoplasts derived from in vitro matured bovine oocytes. The use of cardiomyocytes maintained at 37°C in nuclear transfer, resulted in a significantly reduced proportion of blastocysts compared to adult fibroblasts (14.0% versus 32.7%). Storage of cardiomyocytes at 4°C prior to nuclear transfer was not compatible with blastocyst development. It is expected that this system will be valuable for investigating the reprogramming of gene expression which occurs after somatic cell nuclear transfer.