Priming human fibroblasts with low levels (100 units/ml) of homologous interferon (IFN) prior to induction of gene expression with poly(rI.rC) results in the accumulation of 5-10-fold higher levels of IFN-beta 1 mRNA when compared to induced but nonprimed cells. By run-on transcription assay we have determined that this increase results mainly from increased transcription rate of the IFN-beta 1 gene. We have also shown that priming leads to elevated cytoplasmic steady state levels of two additional RNA species which initiate at the major IFN-beta 1 mRNA initiation site and extend beyond the polyadenylation site for the IFN-beta 1 0.9-kilobase mRNA. Located next to the 3' end of the IFN-beta 1 gene we have also identified a novel poly(rI.rC)-induced gene which like the IFN-beta 1 gene is highly activated in cells primed with IFN prior to poly(rI.rC) induction. However, the expression of another poly(rI.rC)-induced gene located on a different chromosome is only slightly affected by the priming procedure used here. We postulate that IFN priming acts specifically on certain genes rather than by a general potentiation of poly(rI.rC) induction.