Abstract Transcriptomic analyses using high-throughput methods have revealed abundant antisense transcription in bacteria. Antisense transcription is often due to the overlap of mRNAs with long 5′ or 3′ regions that extend beyond the coding sequence. In addition, antisense RNAs that do not contain any coding sequence are also observed. Nostoc sp. PCC 7120 is a filamentous cyanobacterium that, under nitrogen limitation, behaves as a multicellular organism with division of labor among two different cell types that depend on each other, the vegetative CO2-fixing cells and the nitrogen-fixing heterocysts. The differentiation of heterocysts depends on the global nitrogen regulator NtcA and requires the specific regulator HetR. To identify antisense RNAs potentially involved in heterocyst differentiation, we assembled the Nostoc transcriptome using RNA-seq analysis of cells subjected to nitrogen limitation (9 or 24 h after nitrogen removal) in combination with a genome-wide set of transcriptional start sites and a prediction of transcriptional terminators. Our analysis resulted in the definition of a transcriptional map that includes >4,000 transcripts, 65% of which contain regions in antisense orientation to other transcripts. In addition to overlapping mRNAs, we identified nitrogen-regulated noncoding antisense RNAs transcribed from NtcA- or HetR-dependent promoters. As an example of this last category, we further analyzed an antisense (as_gltA) of the gene-encoding citrate synthase and showed that transcription of as_gltA takes place specifically in heterocysts. Since the overexpression of as_gltA reduces citrate synthase activity, this antisense RNA could eventually contribute to the metabolic remodeling that occurs during the differentiation of vegetative cells into heterocysts.