Background: The Old-World sand fly, Phlebotomus papatasi (Scopoli), a vector of zoonotic cutaneous leishmaniasis, is usually controlled by insecticides, including anticholinesterases. Previous studies revealed 85% amino acid sequence identity of recombinant P. papatasi acetylcholinesterase (rPpAChE1) to mosquito AChE and identified synthetic carbamates that selectively inhibited rPpAChE1 and circumvented the G119S mutation responsible for high level resistance to anticholinesterases. This study reports the construction, baculovirus expression, and biochemical properties of rPpAChE1 containing the F290V and F331W orthologous mutations from mosquitoes.Methods: Recombinant PpAChE1 enzymes with or without the F290V, F331W, and G119S orthologous mosquito mutations were expressed in Sf21cells utilizing the baculoviral system. Ellman assays determined changes in catalytic properties and inhibitor sensitivity resulting from wild type and mutant rPpAChE1 containing single or combinations of orthologous mosquito mutations.Results: Each of the orthologous mutations (F290V, F331W, and G119S) from mosquito AChE significantly reduced inhibition sensitivity to organophosphate or carbamate pesticides, and catalytic activity was lost when they were expressed in combination. Novel synthetic carbamates were identified that significantly inhibited the rPpAChEs expressing each of the single orthologous mosquito mutations.Conclusions: These novel carbamates could be developed as efficacious insecticides with improved specificity and safety for use in sand fly or mosquito populations expressing the mutant AChEs.