Staphylokinase is a promising blood-clot dissolving agent for the treatment of patients suffering from a heart attack. It would be desirable to produce this protein in large quantities for biochemical characterization and clinical trials. Production of intact, biologically active staphylokinase from bacterial expression systems has been a challenge because of N-terminal microheterogeneity, plasmid instability, or low-production yield. By using a seven-extracellular-protease deficient Bacillus subtilis strain, WB700, intact staphylokinase can be produced via secretion. However, native staphylokinase gene (sak) in a high-copy number plasmid was found to be unstable in B. subtilis. To optimize the production and the stability of the expression vectors, both the promoter and the signal sequence of sak were replaced by B. subtilis promoters (P43, a constitutively expressed promoter; P-amyr a stationary-phase promoter; and P-sacB' a sucrose-inducible promoter) and the levansucrase-signal sequence, respectively. This overcame the plasmid instability problem. To enhance transcription from the sacB promoter, degQ encoding a transcriptional activator for sacB and other protease genes was also installed in the expression vector. The use of WB700 as the expression host allowed enhanced production of staphylokinase from the sucrose-inducible plasmid without causing any obvious degradation of staphylokinase. Both the P43 and P-sacB (with DegQ) promoters worked well. Over 90% of staphylokinase synthesized can be secreted effectively. With the optimization of both the culture media and the fermentation conditions, production of staphylokinase reached a level of 337 mg/L, and staphylokinase could be purified to homogeneity by a simple three-step purification scheme. Secreted staphylokinase did not show any N-terminal heterogeneity. This presents an attractive system for the production of staphylokinase in both high quality and quantity.