The murine atrial cardiomyocyte-derived cell line HL-1 is a widely accepted tool to investigate cardiomyocte activity. It is assumed that this cell line consists of a homogeneous cell type which allows using them for drug screening experiments. Here we show evidence that there are at least two different cell types. These were grouped into regular cardiomyocytes as well as into pacemaker cells based on their electrophysiological behaviour. Stimulation of the cell layer led to a uniform excitation spread always starting in the pacemaking area, independent of the stimulation location. This indicates that the pacemaker cells are the primary voltage sensing cells.