Abstract Specific primers for nine mouse interferon-alpha (IFN-α) subtypes, namely, IFN-α1, IFN-α1–9, IFN-α2, IFN-α4, IFN-α5, IFN-α7, IFN-α6/8, IFN-α11, and IFN-αB, were designed and evaluated on Poly(I)·Poly(C)-induced and influenza virus-infected L929 cells. Specificity of the primers was confirmed in a cross-polymerase chain reaction (cross-PCR). IFN-α1, IFN-α1–9, IFN-α4, IFN-α6/8, IFN-α11, and IFN-αB were found to be induced in L929 cells 6–9 h after Poly(I)·Poly(C) treatment. The amplification of a particular subtype was not biased in the presence of excess of other templates. Differential expression of the IFN-α subtypes was observed in influenza A/NWS/33- and B/Lee/40-infected L929 cells. A/NWS/33 virus was found to upregulate the gene expression of IFN-α1, IFN-α4, IFN-α6/8, IFN-α11, and IFN-αB in L929 cells as early as 6 h after infection. In B/Lee/40-infected L929 cells, only IFN-α4 was upregulated. Our results suggest that the designed primers will serve as a useful tool in analyzing the expression of IFN-α subtypes in various systems and hence for the evaluation of their function.