We developed a method for quantitating closely related mRNAs by S1 mapping and used it to determine the levels of mRNAs for IFN-beta, IFN-gamma and various alpha IFNs (IFN-alpha 1, -alpha 2, -alpha 4, -alpha 5, -alpha 6, -alpha 7, -alpha 8 and -alpha 14) in human peripheral blood leukocytes, lymphoblastoid (Namalwa), HeLa and human fibroblastic cells, induced in different fashions. The ratio of alpha to beta IFN transcripts varied greatly, depending on the cell type. The levels of the individual IFN-alpha RNAs were very different: IFN-alpha 1, -alpha 2 and -alpha 4 RNAs constituted the major fraction of the IFN-alpha transcripts measured. Moreover, there was a striking difference in the proportion of individual IFN-alpha mRNA species in different cell types. Use of different induction protocols did not significantly affect the proportion of IFN mRNAs. IFN production was not proportional to mRNA level in all cases, as lymphoblastoid cells induced by incubation at high density and virus-induced HeLa cells contained high levels of IFN-beta but produced little antiviral activity.