Nitrogenase activity has been found associated with roots of sweet potato (Ipomoea batatas (L.) Lam). The objective of this investigation was to characterize N2-fixing isolates of the genus Azospirillum found in association with roots of different sweet potato cultivars. Five cultivars ('Carver', 'Jewel', 'Rojo Blanco', 'Centennial', 'Southern Queen') were grown in greenhouse pots on Norfolk sandy loam at pH 6.5 for periods of 1 to 3 months. Enrichment cultures of fibrous and storage root samples were grown on N-free semisolid malate medium at 35 °C. Sixteen bacterial isolates with taxonomic characteristics similar to those of Azospirillum were isolated from the five cultivars. Nitrogenase activities of single colonies grown in semisolid media for 30 h ranged from 32 to 472 nmol C2 H4∙culture−1∙h−1. The isolates were characterized by their acidification of carbon sources, ability to reduce nitrate, and morphology.