Bacterial protein export involves the translocation of precursor proteins across the inner cytoplasmic membrane. Over 100 proteins are exported from Escherichia coli. This study showed that energy in the form of ATP and membrane gradient energy is essential for the export of leucine specific binding protein and (3-lactamase precursors. Ionophores or combinations of ionophores (SF6847, valinomycin/nigericin and valinomycin/monensin) which dissipate protonmotive force inhibit protein export. Valinomycin alone also inhibits export, but not as well as reagents which dissipate protonmotive force. Nigericin or monensin alone slightly stimulate protein export. These results suggest that the transmembrane electrical potential ( DY) is the component of membrane gradient energy necessary for precursor protein export. ATP is necessary for the export of precursors. In the absence of ATP, in vitro export of leucine specific binding protein and B-lactamase precursors is not observed. An upper limit of l0μM was determined for the effective Km for ATP during in vitro protein export. It was also shown that ATP is consumed during the export process. The SecA protein was shown to contain an ATPase activity that is stimulated by the presence of inverted membranes and purified LSBP precursors. Vanadate and diethylstilbestrol, inhibitors of ATPases, inhibit in vitro protein export. Vanadate also inhibits SecA ATPase activity which depends on membranes and precursors. Vanadate is a specific inhibitor of P-type ion translocating ATPases. This study showed primary sequence homology between part of the SecA protein and the phosphorylation sequence of P-type ATPases. Sequence homology, vanadate inhibition of SecA ATPase activity, and vanadate inhibition of in vitro protein export suggest that SecA may function by a mechanism similar to the E1E2 mechanism found in P-type ATPases. Phosphorylation of two proteins with apparent sizes of 62 and 37 kDa is observed to occur in an export-associated fashion. This phosphorylation is dependent on membranes and precursors, is sensitive to hydroxylamine, and is sensitive to inhibitors of protein export, including valinomycin/nigericin and vanadate. Furthermore, phosphorylation of the 62 kDa protein is dependent on the presence of SecA. The phosphate linkage appears to be an acyl phosphate based on hydroxylamine sensitivity and reduction of the acyl phosphate linkage by NaCNBH3. Both proteins appear to be peripherally associated with the cytoplasmic face of the inner membrane, which is also consistent with a possible role in the bacterial protein export process.