Plasmacytoid dendritic cells (pDCs) represent a highly specialized immune cell subset and are considered to be the main sentinels against viral infections and play an important role in the development of immune tolerance. pDCs are able to recognize cytosine-phosphate-guanosine (CpG) motifs within microbial DNA, which are unmethylated CG dinucleotides in a certain sequence context and trigger the secretion of interferon (IFN)-α and other proinflammatory cytokines. Here we used the typical class A CpG oligodeoxynucleotide (ODN) 2216, the B-class ODN 2006, and the newly synthesized CpG ODN TM6₄ to explore the potency and kinetics of IFN-α stimulation of pDC. TM6₄ CpG ODN has a hexanucleotide sequence TCGTGT that leads to an increased cellular uptake and features a CpG nucleotide within the sequence that leads to a potent specific B-cell stimulation, thus characteristics similar to a class B CpG. Our data reveals that all CpGs act as both dosage- and time-dependent stimuli of IFN-α secretion. The relationship between concentration of the stimulant and the secreted amount of IFN-α is not linear and results in a plateau formation, with saturation kinetics. Alteration to the backbone can change duration and quantity of overall IFN-α secretion.