Three strains of A. pullulans showed different ability to produce pullulan in static and shaked cultures.  Shaked cultures produced high amounts (2->5 folds) than static cultures.  A. pullulans ATCC 42023 recorded the highest pullulan accumulation, followed by A. pullulans ATCC 12535.  A. pullulans ATCC 16628 showed feeble pullulan production in spite of its good characteristics.  Production of pullulan was characterized by appearance of sharp peak coinciding with the 3 days' interval of incubation for A. pullulans ATCC 16628 shaked cultures.  For other shaked cultures, the peaks coincided with 6 days.  Pullulan production was highly dependent on the carbon source on which they were cultivated.  Some carbon sources were able to induce pullulan production (2.05–24.18 g/l).  The pH of pullulan producing cultures decreased to a pH range of 2.69-3.00.  A pH range of 5.46–6.29 were recorded in cultures containing sugar cane and sugar beet molasses as carbon and/or energy sources.  Sugar cane molasses or sugar beet molasses, and their treatment methods affected pullulan production.  EDTA treated sugar beet molasses promoted the higher pullulan production (14.83 g/l) for A. pullulans ATCC 12535.  Growing of A. pullulans ATCC 42023 on treated molasses resulted in pullulan production smaller than those produced during growth on glucose syrup.  Growing on molasses as the sole carbon source led to little change in culture pH.  Molasses promoted remarkable growth of pullulan producing strains.Urea+ yeast extract promoted the highest production of pullulan with A. pullulans ATCC 12535, while yeast extract alone appeared as being the most favorable nitrogen source for A. pullulans ATCC 42023.  Na₃PO₄.12H₂O allowed the highest production of the polymer pullulan with A. pullulans ATCC 12535, while Na₂HPO₄ appeared as being the most favorable phosphorus source for A. pullulans ATCC 42023.  The majority of phosphorus sources were not favorable for the test strains.  pH 5.5 seemed to be optimal for pullulan accumulations by the two test strains.  Outside pH 5.5, the pullulan formation was repressed.  Extremes of both acidity and alkalinity adversely affected both the pullulan and biomass production.  5% and 10% inoculum size allowed the highest production of pullulan with A. pullulans ATCC 12535 and A. pullulans ATCC 42023, respectively.  Conversion coefficient (%) at 5% inoculum size reached 59.48% for A. pullulans ATCC 12535 and at 10% inoculum size reached 73.67% for A. pullulans ATCC 42023.  Pullulan yield % at (5%) inoculum size reached 48.60% for A. pullulans ATCC 12535 and at 10% inoculum size reached 66.70% for A. pullulans ATCC 42023.  Pullulan produced has greenish colour, soluble in water at 25°C, the pH for solution soluble in water (from 5-7), maximum ash 2% and melting point over 300°C.  IR was studied for pullulan.