It has been suggested (L. H. Harris, R. P. Desai, N. E. Welker, and E. T. Papoutsakis, Biotechnol. Bioeng. 67:1-11, 2000) that butyryl phosphate (BuP) is a regulator of solventogenesis in Clostridium acetobutylicum. Here, we determined BuP and acetyl phosphate (AcP) levels in fermentations of C. acetobutylicum wild type (WT), degenerate strain M5, a butyrate kinase (buk) mutant, and a phosphotransacetylase (pta) mutant. A sensitive method was developed to measure BuP and AcP in the same sample. Compared to the WT, the buk mutant had higher levels of BuP and AcP; the BuP levels were high during the early exponential phase, and there was a peak corresponding to solvent production. Consistent with this, solvent formation was initiated significantly earlier and was much stronger in the buk mutant than in all other strains. For all strains, initiation of butanol formation corresponded to a BuP peak concentration that was more than 60 to 70 pmol/g (dry weight), and higher and sustained levels corresponded to higher butanol formation fluxes. The BuP levels never exceeded 40 to 50 pmol/g (dry weight) in strain M5, which produces no solvents. The BuP profiles were bimodal, and there was a second peak midway through solventogenesis that corresponded to carboxylic acid reutilization. AcP showed a delayed single peak during late solventogenesis corresponding to acetate reutilization. As expected, in the pta mutant the AcP levels were very low, yet this strain exhibited strong butanol production. These data suggest that BuP is a regulatory molecule that may act as a phosphodonor of transcriptional factors. DNA array-based transcriptional analysis of the buk and M5 mutants demonstrated that high BuP levels corresponded to downregulation of flagellar genes and upregulation of solvent formation and stress genes.