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Functional expression and spectroscopic analysis of luciferases from Lampyris turkestanicus and Photinus pyralis were carried out. cDNA encoding L. turkestanicus luciferase was isolated by reverse transcription‐polymerase chain reaction, cloned, and functionally expressed in Escherichia coli. The luciferases were purified to homogeneity using Ni‐nitrilotriacetic acid Sepharose, and kinetic properties of luciferase from L. turkestanicus were compared with that from P. pyralis. Amino acid differences in its primary structures in relation to P. pyralis luciferase brought about changes in the kinetic properties of the enzyme as evidenced by substantial lowering of K_m for ATP, increased light decay time, and decreased thermostability. Luciferase from L. turkestanicus was used to carry out Michaelis‐Menten kinetics with a K_m of 95.5 μM for ATP and 20 μM for luciferin. Maximum activity was recorded at pH 8.5, so it might be …