Acidic phospholipids have been shown to promote dissociation of bound nucleotides from Mycobacterium tuberculosis DnaA (DnaATB) purified under denaturing conditions [Yamamoto et al., (2002) Modulation of Mycobacterium tuberculosis DnaA protein–adenine-nucleotide interactions by acidic phospholipids. Biochem. J., 363, 305–311]. In the present study, we show that a majority of DnaATB in non-overproducing cells of M. tuberculosis is membrane associated. Estimation of phospholipid phosphorus following chloroform: methanol extraction of soluble DnaATB purified under native conditions (nDnaATB) confirmed the association with phospholipids. nDnaATB exhibited weak ATPase activity, and rapidly exchanged ATP for bound ADP in the absence of any added phospholipids. We suggest that the outcome of intra-cellular DnaATB–nucleotide interactions, hence DnaATB activity, is influenced by phospholipids.