A recently developed mechanistic mathematical model of the bovine estrous cycle was parameterized to fit empirical data sets collected during one estrous cycle of 31 individual cows, with the main objective to further validate the model. The a priori criteria for validation were (1) the resulting model can simulate the measured data correctly (i.e. goodness of fit), and (2) this is achieved without needing extreme, probably non-physiological parameter values. We used a least squares optimization procedure to identify parameter configurations for the mathematical model to fit the empirical in vivo measurements of follicle and corpus luteum sizes, and the plasma concentrations of progesterone, estradiol, FSH and LH for each cow. The model was capable of accommodating normal variation in estrous cycle characteristics of individual cows. With the parameter sets estimated for the individual cows, the model behavior changed for 21 cows, with improved fit of the simulated output curves for 18 of these 21 cows. Moreover, the number of follicular waves was predicted correctly for 18 of the 25 two-wave and three-wave cows, without extreme parameter value changes. Estimation of specific parameters confirmed results of previous model simulations indicating that parameters involved in luteolytic signaling are very important for regulation of general estrous cycle characteristics, and are likely responsible for differences in estrous cycle characteristics between cows.