Abstract: A process for the bacterial production of the hydrocarbon isoprene (2-methyl-1,3-butadiene) is described and claimed. Various bacterial species, both Gram-negative and Gram-positive, were found to produce isoprene, and a method to isolate isoprene-producing bacteria from natural sources is described. Production of isoprene is confirmed by gas chromatography and gas chromatography-mass spectrometry. Of the tested species, Bacillus produces the most isoprene. Growth in a variety of media, including a chemically defined salts medium, in the presence of amino acid sources (casein, gelatin, or lactalbumin hydrolysates) supports isoprene production in logarithmic phase of growth, and with Bacillus cultured in a stirred fermentor, isoprene production occurs both in growing and non-growing cells. High isoprene production rates can also be restored to nutrient-limited cells by adding fresh media, suggesting isoprene production is dependent on nutrient availability.