Lymphocyte activation is initiated by a global increase in messenger RNA synthesis. However, the mechanisms driving transcriptome amplification during the immune response are unknown. By monitoring single-stranded DNA genome wide, we show that the genome of naive cells is poised for rapid activation. In G0, ∼90% of promoters from genes to be expressed in cycling lymphocytes are polymerase loaded but unmelted and support only basal transcription. Furthermore, the transition from abortive to productive elongation is kinetically limiting, causing polymerases to accumulate nearer to transcription start sites. Resting lymphocytes also limit the expression of the transcription factor IIH complex, including XPB and XPD helicases involved in promoter melting and open complex extension. To date, two rate-limiting steps have been shown to control global gene expression in eukaryotes: preinitiation complex assembly and polymerase pausing. Our studies identify promoter melting as a third key regulatory step and propose that this mechanism ensures a prompt lymphocyte response to invading pathogens. Display omitted •Lymphocyte activation induces a proportional amplification of the transcriptome•ssDNA-seq detects promoter melting and non-B DNA in living cells•Promoters in G0 lymphocytes are PolII loaded but unmelted•TFIIH expression and activity are limited in G0 lymphocytes In addition to preinitiation complex assembly and polymerase pausing, DNA melting can also regulate transcription in eukaryotes. In lymphocytes, immune promoters loaded with polymerase are restrained until the DNA is melted, possibly via changes in TFIIH levels.