An IFN-alpha heteroduplex-tracking assay (IFN-HTA) was developed to quantify the frequency of expression of the 16 genes coding for related interferon-alpha (IFN-alpha) subtypes in mice. In mLN of mice treated with Poly (I:C), we observed the induction of three sequential waves of type I IFN production, instead of two as is commonly described: early IFNs after 1 h (IFN-beta), late IFNs after 3 h (mostly IFN-alpha1, -alpha2, -alpha 4 and -alpha 5) and "secondary late IFNs" after 6 h (IFN-alpha 6T and -alpha 8/6). The late IFN wave was associated with the upregulation of the interferon regulatory factor (IRF)-7 mRNA and proteins, whereas the secondary late IFN wave was associated with a slight upregulation of IRF-8 mRNA. Type I IFNs produced in the thymus were associated with a distinct IRF mRNA expression pattern. This IFN-HTA strategy can serve as a useful tool to qualify and quantify the expression of various IFN-alpha subtypes under distinct immune responses and thus provides a first step in evaluating their function.