Abstarct: The present study is aiming to investigate the effect of platooning characteristics on performance measures of two-lane roads baxsed on gap acceptance and rejected gaps of vehicles. Further, platooning characteristics of vehicles in two-lane roads and nonlinear regression traffic flow in order to estimate capacity and level of service (LOS) regarding number of follower per capacity (NFPC) and percent time spent following (PTSF) are examined in the present study. Thus, in order to reach the aims, firstly platooning characteristics are considered as time headway, average travel speed (ATS), number of platoon, percentage of heavy vehicles, PTSF, number of followers, number of overtaking maneuvers, and density in four studied two-lane roads in Gilan and Mazandaran, Iran. Thus, the results of the Pearson correlation analysis for the effect of platooning characteristics on performance measures of two-lane roads indicated that traffic flow has a higher correlation with the average speed of platoon, number of overtaking maneuvers, number of platoon, average travel speed, density, PTSF, percentage of heavy vehicles, and time headway, respectively. Additionally, the effect of platooning characteristics on traffic flow of two-lane roads indicated that by an increase in number of platoon, time headway is decreased which is reached in a critical point as 2.4 seconds. By increasing the number of platoon, ATS is decreased and PTSF is increased in which the number of overtaking maneuvers is increased, accordingly. Moreover, the results of NFPC, and PTSF considering the nonlinear regression traffic flow in comparison with HCM (2010) showed that NFPC proposed in the present study predicted LOS appropriately for unsaturated and saturated traffic conditions in comparison with two performance measure including ATS, and PTSF for class I two-lane roads. Increasing the values of the PTSF up 65% also led to a decrease by 21% in capacity of two-lane roads.