Nitric oxide (NO) plays important roles in the olfactory center of various animals. In the terrestrial slug, NO is indispensable for field potential oscillation in the higher olfactory center, the procerebrum (PC), and also for odor learning. Here we identify a novel NO synthase (NOS) gene, limNOS2, in the terrestrial slug. The mRNA (similar to 10 kb) of limNOS2 encodes a protein consisting of 1616 amino acids, including a PDZ domain. The protein has 70.0% sequence identity with the previously identified limNOS1 gene. In contrast to limNOS1, however, limNOS2 is expressed specifically in the PC. Moreover, most of the cells in the PC contain limNOS2 mRNA, indicating that the nonbursting neurons, the major constituent of the PC, have this mRNA. The expression pattern of limNOS2 conforms well to the pattern of NOS enzymatic histochemical staining. Our present findings indicate that limNOS2 is responsible for most of the NO generation in the PC. (C) 2009 Elsevier Inc. All rights reserved.