Bioinformatic approaches employed to analyse intergenic regions of Pseudomonas aeruginosa O1 (PAO1) for small RNAs (sRNAs) revealed a putative RNA gene encoded upstream of the nitrate assimilation operon nirBD-PA1779-cobA. Here, we show that this RNA, termed nitrogen assimilation leader A (NalA), represents the leader RNA of the nirBD-PA1779-cobA operon, and that nalA transcription is σ(54)- and NtrC-dependent. A PAO1 nalA deletion strain and a strain bearing a deletion in ORF PA1785 failed to grow on nitrate. PA1785 was identified as a homologue of the Azotobacter vinelandii nasT gene, the product of which is required for transcription of the A. vinelandii nitrite/nitrate reductase operon. Collectively, these studies reveal that transcriptional antitermination of the leader RNA NalA is required for expression of the PAO1 nitrate assimilation operon, and that this process is governed by conserved functions in PAO1 and A. vinelandii.