Aim: In this study we investigated the localisation and function of atypical Protein kinase C (PKC) and Protein kinase D (PKD) isoforms in cardiomyocytes. The presence of multiple isoforms of PKC and PKD in cardiomyocytes suggests that individual isoforms mediate unique functions in the heart. Some of these kinases are very well characterised whereas for others little work is done so far. Methods: We created replication incompetent adenoviruses expressing isoforms of the PKC and PKD families that are labeled with fluorescent proteins (FP). Adult rat cardiomyocytes were transduced and the localisation of the protein kinases was studied by confocal microscopy and immunocytochemistry. Using site-directed mutagenesis of PKCiota we created constitutively active or inactive isozymes. When expressed these isozymes are believed to be dominantly active or inactive. Results: Adenoviral gene transfer of such FP-coupled PKC/PKD isoforms into freshly isolated rat ventricular myocytes resulted in robust expression levels at day 2 and 3 after isolation. High resolution confocal microscopy of living myocytes showed highly specific localisation of the PKC to subcellular structures. We confirmed such subcellular distributions by immunocytochemistry of either untransduced or transduced myocytes. When challenged with phorbol esters such as PMA, PKD3 displayed translocation to the plasma-membrane while the atypical PKCiota was unaffected by this treatment. Expression of the dominant negative and positive mutants of PKCiota allowed us to study the effect of the activity level of this isozyme on cellular calcium-signalling in rat cardiac myocytes. Conclusion: This work provides new insights into the localisation and function of so far uncharacterised Protein kinases in the heart.