Sequence determinants that distinguish between promoter and enhancer activities are poorly characterized. Nguyen et al. used massive parallel promoter and enhancer reporter assays in mouse cortical neurons to examine the activities of multiple short (139 bp) sequences that cover hundreds of conserved promoters and enhancers. About 30% of the sequences had activity; the mean enhancer activity of these sequences was similar for promoter-derived and enhancer-derived sequences, but promoter-derived sequences had greater promoter activity than enhancer-derived sequences. This indicates that sequences harbouring strong promoter activity have additional sequence determinants to enhancer sequences. Indeed, the binding motifs of several transcription factor families had both strong promoter and enhancer activity, whereas binding motifs of others had only enhancer activity. Finally, some transcription factors could increase the promoter activity of enhancers.