A five-level-four-factor central composite rotary design was employed to find out the interactive effects of four variables, viz. concentrations of acetate, glucose and K2HPO4, and dark incubation period on poly-beta-hydroxybutyrate (PHB) production in a N-2-fixing cyanobacterium, Nostoc muscorum. Acetate, glucose and dark incubation period exhibited positive impacts on PHB yield. Using response surface methodology (RSM), a second order polynomial equation was obtained by multiple regression analysis. A yield of 45.6% of dry cell weight (dcw) was achieved at reduced level of nutrients, i.e. 0.17% acetate, 0.16% glucose and 5 mg 1(-1) K2HPO4 at a dark incubation period of 9 5 It as compared to 41.6% PHB yield in 0.4% acetate, 0.4% glucose and 40 mg 1(-1) K2HPO4 at a dark incubation period of 168 It under single factor optimization strategy. (c) 2006 Elsevier Ltd. All rights reserved.