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The role of GTP and other ribonucleoside phosphates in the differentiation of Clostridium acetobutylicum and two sporulation-deficient mutants was investigated. The clostridial stage (cls-1) mutant was unable to produce the clostridial stage, solvents, granulose, capsules or endospores, whereas the sporulation-deficient (spo-1) mutant produced the clostridial stage, solvents, granulose and capsules, but did not form a forespore septum. The nucleoside triphosphate profiles of the wild-type and spo-1 mutant were similar and were characterized by a trough in nucleotide levels which occurred just prior to the pH break point, the onset of the stationary growth phase, clostridial stage formation and the transition from the acidogenic to the solventogenic phase. The nucleoside triphosphate concentrations during the exponential growth phase were much lower than those found during the stationary phase. The nucleotide levels in the cls-1 mutant during the exponential phase were comparable to those observed in the wild-type and spo-1 mutant. However, the nucleotide levels in the cls-1 mutant did not increase during the stationary growth phase. The involvement of nucleotide levels, particularly that of GTP, in the differentiation of C. acetobutylicum was indicated by the effect of inhibitors, which have been shown to decrease ribonucleotide levels in other organisms and cause an increase in sporulation.