Aims: To optimize the nutritional and environmental conditions for growth of and poly-beta-hydroxybutyrate (PHB) accumulation in Bacillus mycoides RLJ B-017. Methods and Results: An isolate, identified as B. mycoides, was grown on different sources of carbon and nitrogen. Among these, sucrose, beef extract and di-ammonium sulphate were found to be the most suitable for growth and PHB accumulation. The overall maximum value of PHB (%) in cells, PHB yield (Y-p/s) and productivities (Q(p) and q(p)) were 69.4 +/- 0.4% dry cell weight (DCW), 0.21 g(p) g(s)(-1), 0.104 +/- 0.012 g(p) l(-1) h(-1) and 0.03 g(p) g(x)(-1) h(-1), respectively when grown in a medium containing 20 g(s) l(-1) sucrose, supplemented with di-ammonium sulphate. The addition of beef extract increased the value of PHB (%) in cells, PHB yield and productivities by 17.58 +/- 0.3, 23.8, 19.23 +/- 0.3 and 13.8 +/- 0.2%, respectively. The overall maximum values of PHB (% DCW), PHB yield and productivities were obtained at pH 7.0 +/- 0.1, temperature 30 +/- 0.5degreesC, agitation 650 rev min(-1) and oxygen transfer rate Conclusions: Sucrose, glucose and fructose were found to be more suitable for cell growth and PHB accumulation, but sucrose was less expensive than glucose. Among the nitrogen sources, beef extract and di-ammonium sulphate promoted PHB synthesis. The accumulation of PHB was observed to be growth associated. Significance and Impact of the Study: Gram-positive bacteria have not been reported to accumulate large amounts of polyhydroxyalkanoate and hence have not been considered as potent candidates for industrial production. A number of Bacillus spp. have been reported to accumulate 9-44.5% DCW PHB. By comparison, Bacillus RLJ B-017 contained 69.4 +/-0.4% DCW PHB. Therefore, this strain has been considered as a potent organism for industrial interest. A relatively high yield of PHB was obtained in this wild strain and PHB synthesis was independent of nutrient limitation. The conditions for the higher PHB yield and productivity will be optimized in the next phase using fed-batch culture.