Expression of the heterodimeric cytokine interleukin-(IL-)12 is induced by pattern recognition receptors responding to microbial stimuli such as lipopolysaccharide (LPS) and products of the immune system such as interferon-gamma (IFN-gamma) and CD40L. The formation of bioactive IL-12 requires equimolar synthesis of p35 and p40 subunits. However, p35 expression limits the amount of IL-12 formed. Transcription of the gene for the p35 subunit of IL-12 initiates within the first exon, an alternate first exon (exon 1a), or second exon. Here we show that LPS and IFN-gamma/CD40 ligation increase the amount of total p35 mRNA in splenic adherent cells (SAC) to a similar extent. However, the exon 1 transcript was a smaller fraction of total p35 mRNA in IFN-gamma/CD40-stimulated cells than in unstimulated or LPS-stimulated cells. Despite comparable levels of total p35 mRNA, LPS-induced p35 exon 1 transcripts led to significantly more bioactive IL-12 from SAC than IFN-gamma/CD40-induced exon 1a/exon 2 transcripts as measured by ELISA. The data suggest that LPS-inducible p35 synthesis from exon 1 p35 transcripts leads to greater amount of bioactive IL-12 than IFN-gamma/CD40-induced p35 expression from alternate p35 exon 1a/exon 2 transcripts.