We evaluated phytohormone and polyamine biosynthesis, siderophore production, and phosphate solubilization in two strains (Cd and Az39) of Azospirillum brasilense used for inoculant formulation in Argentina during the last 20 years. Siderophore production and phosphate solubilization were evaluated in a chemically defined medium, with negative results. Indole 3-acetic acid (IAA), gibberellic acid (GA(3)), and abscisic acid (ABA) production were analyzed by gas chromatography-mass spectrometry. Ethylene, polyamine, and zeatin (Z) biosynthesis were determined by gas chromatography-flame ionization detector and high performance liquid chromatography (HPLC-fluorescence and -UV), respectively. Phytohormones IAA, Z, GA(3), ABA, ethylene, and growth regulators putrescine, spermine, spermidine, and cadaverine (CAD) were found in culture supernatant of both strains. IAA, Z, and GA(3) were found in all two strains; however, their levels were significantly higher (p < 0.01) in Cd (10.8, 2.32, 0.66 microg ml(-1)). ABA biosynthesis was significantly higher (p < 0.01) in Az39 (0.077 microg ml(-1)). Ethylene and polyamine CAD were found in all two strains, with highest production in Cd cultured in NFb plus L-methionine (3.94 ng ml(-1) h(-1)) and Az39 cultured in NFb plus L-lysine (36.55 ng ml(-1) h(-1)). This is the first report on the evaluation of important bioactive molecules in strains of A. brasilense as potentially capable of direct plant growth promotion or agronomic yield increase. Az39 and Cd showed differential capability to produce the five major phytohormones and CAD in chemically defined medium. This fact has important technological implications for inoculant formulation as different concentrations of growth regulators are produced by different strains or culture conditions.