Many mammalian genes are transcribed not only in the forward but also the reverse orientation; yet it is unclear what genetic elements and chromatin features mediate the initiation of such 'divergent' transcription. By mapping genome-wide transcription initiation in HeLa cells using 5′-GRO-seq, Duttke et al. identified multiple divergent promoter pairs that were associated with genes in the forward direction and with non-annotated regions in the reverse direction, and separated by nucleosome free regions (NFRs) of ∼200 bp. In vitro transcription experiments revealed unidirectional transcription from reverse-direction start sites, which was driven by core promoters similar to but not overlapping those driving forward transcription. Importantly, genomic data analyses suggested that about half of HeLa NFR-associated promoters are unidirectional, and thus that divergent transcription is not a ubiquitous feature of mammalian promoters.