The natural volatile hydrocarbon isoprene (2-methyl-l, 3-butadiene) is an essential chemical for polymers, drugs and rubber industry. It is produced naturally by animals, plants and bacteria. The isoprene synthase gene was identified in plants. However, it was not identified in bacteria. Moreover, it was found that Bacillus sp.produced the most isoprene compared to other bacteria species. In this work, the quantitative analysis of isoprene produced by Bacillus subtilis ATCC 6633 and Bacillus stearothermophilus 12980 ATCC was measured using gas chromatography-mass spectrometry (GC-MS) compared to standard isoprene, in which retention time was 3.61 for isoprene standard, 3.69 for B. stearothermophilus and 3.68 for B. subtilis. Additionally, Bacillus stearothermophilus 12980 ATCC produced 1.169 mg/ml isoprene, when incubated at 55^oC for 18h and Bacillus subtilis 6633 ATCC produced 1.071 mg/ml isoprene when incubated at 45^oC for 18h. The synthetic kudzu isoprene synthase gene was used to transform the E. coli DH5α cells.  SDS-PAGE of proteins from the transformed E. coli cells showed a band in the predicted size of the kIspS protein at 65 Kd. In conclusion, from the previous work further studies might develop recombinant bacteria as a biological source capable of isoprene production for chemical feedstock and rubber synthesis.