Background Carbon disulfide (CS2) is a commonly used organic solvent. Many epidemiological investigations and animal experiments have indicated that learning and memory ability can be affected to different degrees after long-term exposure to CS2, but the mechanisms are still unclear. The aim of this study was to explore the possible mechanisms of CS2-related impairment of the learning and memory ability of rats, by investigating the effects of CS2 on nitric oxide synthase (NOS) activity and NOS mRNA expression in rat hippocampus. Methods Rat models of toxicity were generated by inhalation of various doses of CS2. After two months of inhaling intoxication, the activities of constitutive NOS (cNOS) and induced NOS (iNOS) in the hippocampus were measured. The levels of neuronal NOS (nNOS) mRNA and iNOS mRNA were measured by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Results cNOS activity was significantly decreased compared with controls, while iNOS activity was changed only slightly. CS2 treatment significantly decreased nNOS mRNA levels, iNOS mRNA levels were significantly increased only at higher doses of CS2. Conclusion The effect of CS2 on learning and memory ability in rats is related to the activity of NOS and the expression of nNOS in the hippocampus.