Among the different vaccination approaches, DNA/RNA vaccination represents a promising means in particular for the induction of effective cellular immune responses conferred by CD8-positive T lymphocytes. To achieve such immune responses, there is a need for novel delivery systems that allow the introduction of nucleic acids to the cytosol of immune cells. We show, for the first time, the delivery of functional DNA and messenger RNA (mRNA) to mammalian antigen-presenting cells, including murine macrophages and human dendritic cells, using the yeast Saccharomyces cerevisiae as the delivery vehicle. After transfer of the particular nucleic acid, subsequent antigen processing and presentation were demonstrated in a human system. Remarkably, release of DNA/mRNA does not require additional ‘helper’ proteins such as listeriolysin. In conclusion, the yeast-based system described here is superior to many bacterial and viral systems in terms of efficacy, safety and targeting suggesting ‘mycofection’ as a promising approach for the development of a novel type of live vaccines.