Background: Nitric oxide (NO) is implied in many important biological processes in all metazoans from porifera to chordates. In the cuttlefish Sepia officinalis NO plays a key role in the defense system and neurotransmission. Results: Here, we detected for the first time NO, NO synthase (NOS) and transcript levels during the development of S. officinalis. The spatial pattern of NO and NOS is very dynamic, it begins during organogenesis in ganglia and epithelial tissues, as well as in sensory cells. At later stages, NO and NOS appear in organs and/or structures, including Hoyle organ, gills and suckers. Temporal expression of NOS, followed by real-time PCR, changes during development reaching the maximum level of expression at stage 26. Conclusions: Overall these data suggest the involvement of NO during cuttlefish development in different fundamental processes, such as differentiation of neural and nonneural structures, ciliary beating, sensory cell maintaining, and organ functioning. Developmental Dynamics 241:390–402, 2012. © 2011 Wiley Periodicals, Inc.