A complex mixture of methyl-branched alkyl-substituted pyrazines was found in the growth medium of the polymyxin-producing bacterium Paenibacillus polymyxa, and of these, seven are new natural compounds. A total of 19 pyrazine metabolites were identified. The dominant metabolite was 2,5-diisopropylpyrazine as identified using a combination of high-resolution mass spectrometry, 1H- and 13C-nuclear magnetic resonance, gas chromatography-mass spectrometry as well as co-elution with an authentic standard. Its biosynthesis was correlated with growth and production was strongly stimulated by valine supplementation. The other pyrazine metabolites, all related pyrazines with either one, two or three alkyl substituents, were identified by means of their mass spectral data and/or co-elution with authentic standards.