During transcription initiation in vitro, prokaryotic and eukaryotic RNA polymerase (RNAP) can engage in abortive initiation—the synthesis and release of short (2 to 15 nucleotides) RNA transcripts—before productive initiation. It has not been known whether abortive initiation occurs in vivo. Using hybridization with locked nucleic acid probes, we directly detected abortive transcripts in bacteria. In addition, we show that in vivo abortive initiation shows characteristics of in vitro abortive initiation: Abortive initiation increases upon stabilizing interactions between RNAP and either promoter DNA or sigma factor, and also upon deleting elongation factor GreA. Abortive transcripts may have functional roles in regulating gene expression in vivo.