Aims: To isolate the novel nonmelanin pullulan-producing fungi from soil and to optimize the physico-chemical and nutritional parameters for pullulan production. Methods and Results: A selective enrichment method was followed for the isolation, along with development of a suitable medium for pullulan production, using shake flask experiments. Pullulan content was confirmed using pure pullulan and pullulanase hydrolysate. Eurotium chevalieri was able to produce maximum pullulan (38 +/- 1 center dot 0 g l-1) at 35 degrees C, pH 5 center dot 5, 2 center dot 5% sucrose, 0 center dot 3% ammonium sulfate and 0 center dot 2% yeast extract in a shake flash culture medium with an agitation rate of 30 rev min-1 for 65 h. Conclusions: The novel pullulan-producing fungus was identified as E. chevalieri (MTCC no. 9614), which was able to produce nonmelanin pullulan at from poorer carbon and nitrogen sources than Aureobasidium pullulans and may therefore be useful for the commercial production of pullulan. Significance and Impact of the Study: Eurotium chevalieri could produce pullulan in similar amounts to A. pullulans. Therefore, in future, this fungus could also be used for commercial pullulan production, because it is neither polymorphic nor melanin producing, hence its handling during pullulan fermentation will be easier and more economical.