The inhibitory effect of ATP on phosphodiesterase activity plays an important regulatory role in animal cells (Cheung, 1966), but this condition has not been demonstrated in micro-organisms. This communication shows the inhibitory effect of ATP and other purine and pyrimidine trinucleotides on phosphodiesterase activity in Mycobacterium smegmatis. Although trinucleotides inhibit phosphodiesterase activity, mononucleotides or adenosine stimulate enzyme activity. The results from this study suggest that the intracellular concentration of cyclic AMP is influenced by nucleotides, nucleosides and deoxynucleotides.