Transcription of DNA is a fundamental process in all cellular organisms. The enzyme responsible for transcription, RNA polymerase, is conserved in general architecture and catalytic function across the three domains of life. Diverse mechanisms are used among and within the different branches to regulate transcription initiation. Mechanistic studies of transcription initiation in bacteria are especially amenable because the promoter recognition and melting steps are much less complicated than in eukaryotes or archaea. Also, bacteria have critical roles in human health as pathogens and commensals, and the bacterial RNA polymerase is a proven target for antibiotics. Recent biophysical studies of RNA polymerases and their inhibition, as well as transcription initiation and transcription factors, have detailed the mechanisms of transcription initiation in phylogenetically diverse bacteria, inspiring this Review to examine unifying and diverse themes in this process.