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Toll plazas are one of the critical components of a roadway system for capital financing, infrastructure maintenance revenue, or traffic maintenance and congestion control strategies. At the same time, they are among the most complex road structures, as drivers are exposed to a large amount of information and have a short amount of time to make a decision. Since the advent of electronic toll collection (ETC) technology, the complexity of toll plazas has greatly increased. The objective of this study is to investigate the effect of toll plaza design and traffic conditions on drivers’ behavior and level of safety. This study contains two approaches: (1) a microsimulation study using VISSIM and the Surrogate Safety Assessment Model (SSAM); and (2) a driving simulation study. The microsimulation model was calibrated and validated using traffic data from recorded video at the West Springfield toll plaza, Massachusetts, connecting Interstate 90 to Interstate 91 and State Route 5. Distribution of traffic volumes, stop delays at cash lanes, and reduced speed distribution at electronic toll collection (ETC) lanes were used as vii calibration variables, and the number of conflicts was used as a validation parameter. Results identified that the safest lane configuration was the one consisting of only ETC lanes, and the second-safest configurations were the ones that grouped ETC lanes and separated them from cash lanes. In the second part of the study, a simulation model of the same toll plaza was created to be used in a fixed-base driving simulator with a 150 degree field of view. The objective of this part of the study was to investigate drivers’ behavior when they are exposed to different lane configurations and traffic conditions at toll plazas. Independent variables of this study were lane configuration (i.e., which lanes were signed as “E-ZPass” and “Cash”), origin-destination of the subject vehicle (i.e., right or left origin ramp, right or left destination ramp), traffic queue (i.e., having a queue or not), traffic composition (i.e., having a leading heavy vehicle or not), and customer type (i.e., cash or E-ZPass). The result of this simulation study was expected to give a better understanding of drivers’ behavior at toll plazas and might lead to safer toll plaza designs.