In order to elucidate the mechanism of the accumulation of considerable amounts of free nicotinic acid (NA) in the culture medium of Clostridium butylicum, this organism was investigated with regard to its ability to metabolize nicotinamide adenine dinucleotide (NAD) and its immediate biosynthetic precursors, nicotinic acid mononucleotide (NAMN) and nicotinic acid adenine dinucleotide (deamido-NAD). Cell-free extracts of C. butylicum were found to degrade NAMN and deamido-NAD to NA. NAMN, in the presence of adenosine triphosphate (ATP), was converted to deamido-NAD, but only at high concentrations of ATP (20 mM) was significant synthetic activity observed in competition with NAMN degradation. Degradation of both NAMN and deamido-NAD was activated by ATP at concentrations of 5 and 10 mm. Anaerobiosis markedly enhanced the degradation of the nucleotides. The data indicate that the synthesis of NAMN and deamido-NAD prevails over their degradation only in the presence of high concentrations of ATP. NAD was degraded to nicotinamide mononucleotide (NMN) by a pyrophosphatase. Phosphate markedly inhibited both the deamido-NAD and NAD pyrophosphatases. Under anaerobic conditions there was practically no further degradation of NMN to NA, whereas barely measurable amounts of NA were formed under aerobic conditions. All of these observations suggest that, under the given conditions of anaerobiosis and physiological phosphate concentrations, there is very little degradation of NAD to NMN and practically no degradation to NA by C. butylicum. Thus, NAD represents an insignificant source of the NA accumulated in the culture medium. The intermediates in the biosynthetic pathway (NAMN and deamido-NAD) have been shown to be the major source of the NA which is accumulated by C. butylicum.