Gas phase and particulate phase radicals in mainstream cigarette smoke were determined Electron Spin Resonance(ESR) spectroscopy. The free radicals in particulate phase have been investigated by benzene extract of Cambridge Filter Pad containing the smoke condensate. Spin trapping method in conjunction with ESR was used to investigate free radicals in the gas phase of cigarette smoke. Several analytical experiments were conducted in order to determine the optimal conditions for maximum signal intensities and reproducibility of results. All the tests were optimized and normalized using the University of Kentucky 2R4F reference cigarette. The optimal conditions were 0.6 mL for analysis volume of ESR, $4{sim}5;mL$ for collection volume of spin-adducts, and PBN for quantification of free radicals in gas phase. The radical levels of Kentucky 2R4F cigarettes were found $2.18{ imes}10^{14};spins/cig.$ and $2.10{ imes}10^{15};spins/cig.$ in gas phase.