Bacterial PII proteins, encoded by glnB genes, are central signalling molecules in nitrogen regulatory pathways and are modulated by post-translational modification in response to the cellular nitrogen status. The glnB gene was cloned from the filamentous heterocyst-forming cyanobacterium Nostoc punctiforme strain ATCC 29133 (PCC 73102) by heterologous hybridization to a Synechococcus sp. strain PCC 7942 gene fragment. Expression of the cloned gene was verified by hybridization to N. punctiforme total RNA and a single cross-reactive polypeptide was observed in immunoblots of N. punctiforme extracts probed with anti-Synechococcus 7942 PII antiserum. Modification of the purified N. punctiforme PII protein by a Synechococcus 7942 PII kinase was observed, but modified forms of PII were not detected in extracts of N. punctiforme from a variety of incubation conditions. The N. punctiforme glnB gene could not be disrupted by targeted gene replacement unless a second copy of glnB was provided in trans, suggesting that the gene or gene product is essential for growth under the conditions tested.