Although recent neuroimaging studies suggest that prefrontal cortex (PFC) is involved in working memory (WM), the relationship between PFC activity and memory load has not yet been well-described in humans. Here we use functional magnetic resonance imaging (fMRI) to probe PFC activity during a sequential letter task in which memory load was varied in an incremental fashion. In all nine subjects studied, dorsolateral and left inferior regions of PFC were identified that exhibited a linear relationship between activity and WM load. Furthermore, these same regions were independently identified through direct correlations of the fMRI signal with a behavioral measure that indexes WM function during task performance. A second experiment, using whole-brain imaging techniques, both replicated these findings and identified additional brain regions showing a linear relationship with load, suggesting a distributed circuit that participates with PFC in subserving WM. Taken together, these results provide a “dose–response curve” describing the involvement of both PFC and related brain regions in WM function, and highlight the benefits of using graded, parametric designs in neuroimaging research.