The large family of human type I interferon (IFN) includes 13 distinct subtypes of IFN-alpha, all utilizing a single type I IFN receptor. Many viruses have created evasion strategies to disable this cytokine family, highlighting their importance in antiviral defense. It is unclear what advantage the presence of so many different IFN-alpha subtypes provides, but functional differences observed among individual IFN-alpha subtypes suggested that they might play distinct regulatory roles during an immune response. To determine whether IFN-alpha subtype responses differ depending on a particular type of insult and thus whether IFN-alpha subtype responses are flexible to adapt to distinct pathogen challenges, we developed a novel nested multiplex reverse transcriptase polymerase chain reaction assay with which we measured expression of all IFN-alpha subtypes by freshly isolated human plasmacytoid dendritic cells (pDCs), a main source of IFN-alpha following pathogen challenge. Collectively our data show a remarkable stability in the relative magnitude and the kinetics of induction for each IFN-alpha subtype produced by pDC. Although various stimuli used, A-, B- and C-class CpGs, live and heat-inactivated influenza viruses and the TLR7 agonist R837 affected the overall magnitude of the response, each IFN-alpha subtype was induced at statistically similar relative levels and with similar kinetics, thereby revealing a great degree of rigidity in the IFN-alpha response pattern of pDC. These data are most consistent with the induction of optimized ratios of IFN-alpha subtypes, each of which may have differing signaling properties or alternatively, a great degree of redundancy in the IFN-alpha response.