Specific primers for nine mouse interferon-alpha (IFN-alpha) subtypes, namely, IFN-alpha1, IFN-alpha1-9, IFN-alpha2, IFN-alpha4, IFN-alpha5, IFN-alpha7, IFN-alpha6/8, IFN-alpha11, and IFN-alphaB, 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-alpha1, IFN-alpha1-9, IFN-alpha4, IFN-alpha6/8, IFN-alpha11, and IFN-alphaB 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-alpha 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-alpha1, IFN-alpha4, IFN-alpha6/8, IFN-alpha11, and IFN-alphaB in L929 cells as early as 6 h after infection. In B/Lee/40-infected L929 cells, only IFN-alpha4 was upregulated. Our results suggest that the designed primers will serve as a useful tool in analyzing the expression of IFN-alpha subtypes in various systems and hence for the evaluation of their function.