Abstract: Periplasmic ALP from Rhizobium bacteria that catalyzes the efficient hydrolysis of monophosphate ester at alkaline pH was isolated and partially purified. Periplasmic ALP activities have been isolated upon lysozyme (10 mg lysozyme/1 mL Tris-HCl buffer, 30 mM, pH 8.0) treatment of bacterial culture. Based on p-nitrophenyl phosphate as substrate, periplasmic ALP has shown most of the activity in the pH range 6.8 to 11.8 with peak value at pH 9.8. Michaelian kinetic parameters of the enzyme were as follows: Vmax = 10.91 μmol min-1 and Km = 58 μM. The enzyme had a temperature optimum of 25-65°C and was stable between 0-55°C up to 48 h. Maximum periplasmic ALP activity was obtained by 40% (NH4)2SO4 saturation. The ALP activities was affected by metal ions (Ca2+, Mg2+, Co2+ and K+) and inhibitors (sodium fluoride, sodium arsenate, EDTA and THB). Ca2+ and K+ enhanced the ALP activities. Sodium fluoride and sodium arsenate caused drastic inhibition in ALP activity as compared to those of metal chelators (EDTA) and THB (tetra hydro borate). The overall results of the study concluded that Rhizobium ALP were similar to that of E. coli ALP.