Many eyewitness researchers have argued for the application of a sequential alternative to the traditional simultaneous lineup, given its role in decreasing false identifications of innocent suspects (sequential superiority effect). However, Ebbesen and Flowe (2002) have recently noted that sequential lineups may merely bring about a shift in response criterion, having no effect on discrimination accuracy. We explored this claim, using a method that allows signal detection theory measures to be collected from eyewitnesses. In three experiments, lineup type was factorially combined with conditions expected to influence response criterion and/or discrimination accuracy. Results were consistent with signal detection theory predictions, including that of a conservative criterion shift with the sequential presentation of lineups. In a fourth experiment, we explored the phenomenological basis for the criterion shift, using the remember-know-guess procedure. In accord with previous research, the criterion shift in sequential lineups was associated with a reduction in familiarity-based responding. It is proposed that the relative similarity between lineup members may create a context in which fluency-based processing is facilitated to a greater extent when lineup members are presented simultaneously.