With the recent widespread deployment of intelligent transportation systems (ITS) in North America there is an abundance of data on traffic systems and thus an opportunity to use these data in the calibration of microscopic traffic simulation models. Even though ITS data have been utilized to some extent in the calibration of microscopic traffic simulation models, efforts have focused on improving the quality of the calibration based on aggregate form of ITS data rather than disaggregate data. In addition, researchers have focused on identifying the parameters associated with car-following and lane-changing behavior models and their impacts on overall calibration performance. Therefore, the estimation of the Origin-Destination (OD) matrix has been considered as a preliminary step rather than as a stage that can be included in the calibration process. This research develops a methodology to calibrate the OD matrix jointly with model behavior parameters using a bi-level calibration framework. The upper level seeks to identify the best model parameters using a genetic algorithm (GA). In this level, a statistically based calibration objective function is introduced to account for disaggregate form of ITS data in the calibration of microscopic traffic simulation models and, thus, accurately replicate dynamics of observed traffic conditions. Specifically, the Kolmogorov-Smirnov test is used to measure the "consistency" between the observed and simulated travel time distributions. The calibration of the OD matrix is performed in the lower level, where observed and simulated travel times are incorporated into the OD estimator for the calibration of the OD matrix. The interdependent relationship between travel time information and the OD matrix is formulated using a Extended Kalman filter (EKF) algorithm, which is selected to quantify the nonlinear dependence of the simulation results (travel time) on the OD matrix. The two test sites are from an urban arterial and a freeway in Houston, Texas. The VISSIM model was used to evaluate the proposed methodologies. It was found that that the accuracy of the calibration can be improved by using disaggregated data and by considering both driver behavior parameters and demand.