Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family critical for neuronal cell survival and differentiation, with therapeutic potential for the treatment of neurological disorders and spinal cord injuries. The production of recombinant, bioactive BDNF is not practical in most traditional microbial expression systems because of the inability of the host to correctly form the characteristic cystine-knot fold of BDNF. Here, we investigated Brevibacillus choshinensis as a suitable expression host for bioactive BDNF expression, evaluating the effects of medium type (2SY and TM), temperature (25 and 30 A degrees C), and culture time (48-120 h). Maximal BDNF bioactivity (per unit mass) was observed in cultures grown in 2SY medium at extended times (96 h at 30 A degrees C or > 72 h at 25 A degrees C), with resulting bioactivity comparable to that of a commercially available BDNF. For cultures grown in 2SY medium at 25 A degrees C for 72 h, the condition that led to the greatest quantity of biologically active protein in the shortest culture time, we recovered 264 mu g/L of BDNF. As with other microbial expression systems, BDNF aggregates did form in all culture conditions, indicating that while we were able to recover biologically active BDNF, further optimization of the expression system could yield still greater quantities of bioactive protein. This study provides confirmation that B. choshinensis is capable of producing biologically active BDNF and that further optimization of culture conditions could prove valuable in increasing BDNF yields.