This study includes the first systematic comparison of an organic nitrate with the corresponding organic nitrite-isobutyl nitrate and isobutyl nitrite. The spasmolytic activity of the nitrite on isolated rabbit aortic strips was stronger, more rapid in onset, but less stable than the activity of the nitrate. In vitro tolerance to glyceryl trinitrate and isobutyl nitrite greatly weakened the activity of isobutyl nitrate, respectively, but had much less effect on isobutyl nitrite. Possible reasons for these differences are discussed.