We investigated the role of nitric oxide (NO) in learning and memory formation in a radial maze test, by using the NO synthase (NOS) inhibitors, NG-nitro-L-arginine methyl ester (L-NAME) and 7-nitroindazole (7-NI), and an NO precursor, L-arginine. Rats were trained on a working and reference memory task in an eight arm radial maze for 30 days, wherein the same four arms were baited for each daily training trial. Daily administration of the specific neuronal NOS inhibitor 7-NI, as well as the non-selective NOS inhibitor L-NAME, impaired the acquisition of the task. Analysis of memory categories affected by NOS inhibitors revealed that reference memory formation was impaired by the treatment with L-NAME and 7-NI. L-NAME also impaired working memory although 7-NI had no effect. L-Arginine significantly increased the choice accuracy, by reducing the reference memory errors, of the radial arm maze task during the last ten days although it had no effect during the first 20 trials. These results suggest that NO plays an important role in spatial reference memory formation.