Serological relationships amongst different genera and also different species of nonsymbiotic N2-fixing bacteria were studied using Enzyme-linked Immunosorbent Assay (ELISA). Azospirillum lipoferum strains ICM 1001 and 4ABL were serologically distinct from strains of A. brasilense & A. amazonense; however, all of 10 A. lipoferum strains showed weak cross reactions, suggesting that these strains shared some common antigens with the A. lipoferum strains. Antisera of A. brasilense strains (SP 7a, SL 33, and SM 6M) did not cross react with 12 strains of A. lipoferum, 4 strains of A. amazonense, nor with 17 strains of A. brasilense tested. An exception was the B 25 strain, which showed the same reaction as that of homologous antigen against SP 7a antiserum. Antisera of azospirilla did not cross react with other genera of N2-fixing bacteria tested using ELISA. Antiserum of Azotobacter chroococcum (ICM 2001) was genus and species specific. Enterobacter cloacae antiserum was genus specific but strains of E. cloacae and E. aerogenes shared some common antigens. The use of the ELISA to enumerate azospirilla in peat inoculants and broth culture and A. chroococcum in broth culture was investigated. A minimum of 10² and 104 cells of Azotobacter and Azospirillum, respectively, are required for a detectable ELISA reaction. Heat killed cells interfere with the ELISA reaction, limiting the use of this technique when the number of bacterial cells is low and all the cells in a sample are killed
