When individuals acquire new skills, initial performance is typically better and tasks are judged to be easier when the tasks are segregated and practiced by block, compared to when different tasks are randomly intermixed in practice. However, subsequent skill retention is better for a randomly practiced group, an effect known as contextual interference (CI). The present study examined the neural substrates of CI using functional magnetic resonance imaging (fMRI). Individuals learned a set of three 4-element sequences with the left hand according to a block or random practice schedule. Behavioral retest for skill retention confirmed the presence of a typical CI effect with the random group outperforming the block group. Using a go/no-go fMRI paradigm, sequence preparation during the premovement study period was separated from movement execution. Imaging data for the two groups were compared for the first 1/3 and final 1/3 of training trials. Toward the end of training, behavioral performance between the two groups was similar, although the random group would later display a performance advantage on retention testing. During study time, the random group showed greater activity in sensorimotor and premotor regions compared to the block group. These areas are associated with motor preparation, sequencing, and response selection. This pattern of recruitment is consistent with the hypothesis that CI benefits in a sequencing task are due to improved capacity to actively prepare motor responses.