The purpose of the current study was to examine the role of familiarity and lineup procedure on eyewitness identification accuracy. Familiarity was manipulated wherein adolescent participants (N = 623): (1) met with and directly interacted with a confederate, (2) indirectly interacted with a confederate, or (3) did not meet a confederate, before they viewed a crime video in which the confederate was the perpetrator. Three commonly used lineup procedures (i.e., simultaneous, sequential, and elimination-plus) were used, and the presence of the target also was manipulated. Overall, familiarity and lineup procedure impacted identification accuracy, such that in target-present lineups, witnesses were more likely to make a correct identification when they were more familiar (i.e., had direct interaction) with the perpetrator and the sequential procedure was used. Furthermore, in target-absent lineups, witnesses were more likely to make a correct rejection when they were more familiar (i.e., had direct interaction) with the perpetrator and the simultaneous or elimination-plus procedure was used. Taken together, these findings suggest that familiarity, in terms of having a direct interaction with a perpetrator before the commission of a crime, positively influences identification accuracy. Furthermore, these findings provide new, important information regarding the simultaneous-sequential debate and the utility of commonly used lineup procedures when the witness is familiar with the perpetrator.