Infection by influenza A virus has previously been shown to prime macrophages for a high TNF-alpha production. Influenza A virus induced a TNF-alpha mRNA accumulation that consisted of two types: a regular 1.7 kb and an additional high m.w. 2.4 kb species in murine macrophages, and a high m.w. 3.6 kb species in human monocytes. In this study, we further characterized this virus-induced, novel high m.w. TNF-alpha mRNA. The additional high m.w. TNF-alpha mRNA represented a true polyadenylated mRNA and its induction required exposure to infectious viruses. The regular and the high m.w. TNF-alpha mRNA were both found in the nuclear fraction and the cytoplasm. We excluded that the novel high m.w. TNF-alpha mRNA was an intron-containing precursor TNF-alpha mRNA that could have persisted in virus-infected macrophages. When TNF-alpha exons 1 to 4 and TNF-alpha exons 2 to 4 were amplified by polymerase chain reaction, only regular and no high m.w. bands were detected. By use of specific TNF-alpha intron I and intron III cDNA we could definitely demonstrate the absence of introns in the high m.w. TNF-alpha mRNA. The high m.w. TNF-alpha mRNA was free of TNF-beta and TNF intergenic region elements but contained the 5' and 3' untranslated region of TNF-alpha. Influenza A virus infection also induced a double band of IL-1 beta and IL-6 mRNA. Whether this novel high m.w. TNF-alpha mRNA represents a virus-induced abnormality or a superinduction of an otherwise normal but minor TNF-alpha transcript, and whether this high m.w. TNF-alpha mRNA species codes for a biologically active product, remains to be examined.