Background: Acid phosphatase (ACP) is suggested to be one of the virulence factors in leishmania and a correlation has been reported between Leishmanial acid phosphatase (ACP) activity and the severity of the disease in all types of leishmaniasis including zoonotic cutaneous leishmaniasis (ZCL), which is a polymorphic disease. Objective: Characterization of leishmania and its correlation with the severity of the skin lesions in zoonotic cutaneous leishmaniasis. Methods: Promastigotes were isolated from 30 patients with scaly flat ulcers (LP1), volcano-shaped lesions (LP2) and papular forms (LP3) of ZCL. The Km and Vmax values of ACP in the supernatant of lysed promastigotes were calculated in presence and absence of the enzyme inhibitors, fluoride, tartarate, phosphate and salicylate. Results: The Michaelis-Menten substrate saturation kinetics exhibited Km values of 12.5, 16.0, 26.6 mM and Vmax values of 8.78, 5.26 and 1.51 mM/min/mg protein for LP1, LP2 and LP3, respectively. In all isolates, the ACP was inhibited by all four inhibitors but the percentage inhibition was different and each inhibitor showed a particular inhibition pattern for each promastigote preparation. ACP content of LP1 was more sensitive to all four inhibitors and fluoride was more potent inhibitor of the enzyme in three isolates. Conclusion: The differences in ACP kinetics may be interpreted as being consistent with the severity of the skin lesions in Leishmania major infection.