1. 1. Study has been made of the effects of a variety of metabolic inhibitors and divalent cations (Ni 2+ and Mn 2+), normally after 5 min exposure, on the biphasic uptake of inorganic phosphate (P i) exhibited by phosphate-deprived cells of Escherichia coli, strains AB3311 (Reeves met − ) and CBT302 (a ( Ca 2++ Mg 2+)- ATPase-deficient mutant). 2. 2. In AB3311 cells cyanide (1–10 mM) produced comparable reductions in phosphate uptake to anaerobiosis, but in both instances significant uptake was maintained. Examination of intracellular P i concentrations showed that, despite these inhibitions, P i is still concentrated 130 times compared to 394 times under aerobic conditions. Arsenate (100 μM) and iodoacetate (100 μM pre-exposed 15 min) both abolished anaerobic-supported uptake. Under aerobic conditions the former eliminated primary uptake while the latter reduced both phases of uptake 60%. The uncouplers, dinitrophenol (100–1000 μM) and carbonyl cyanide m- chlorophenyl hydrazone (CCCP) (50 μM) produced very significant, but not complete inhibitions of both phases of uptake. Inhibitions by iodoacetate and dinitrophenol were additive while dithiothreitol protected against the effects of 50–250 μm CCCP. N,N′- Dicyclohexylcarbodiimide (DCCD), the potent inhibitor of membrane-bound ( Ca 2+ + Mg 2+)- ATPase , at 10 −3 M caused significant inhibitions of aerobic- (approx. 60%) and anaerobic- (approx. 80%) supported uptakes thus suggesting some obligatory requirement for this ATPase. 3. 3. CBT302 cells, like AB3311, supported P i transport both aerobically and anaerobically, CCCP (50 μM) reduced the primary uptake similarly to AB3311 cells, but the secondary uptake was less affected. DCCD ( 10 −5−10 −3 M ), as expected, showed no effects in contrast to AB3311 cells. 4. 4. In AB3311 cells Ni 2+ (10 mM) caused significant but different reductions of secondary (70%) and primary (33%) phases of phosphate uptake. Mn 2+ (10 mM) showed a greater differential effect with the primary uptake being minimally affected.