The aim of the present study was to characterize the functional relationships between behaviorally evoked regional brain activation and cardiac autonomic activity in humans. Concurrent estimates of regional cerebral blood flow (rCBF; obtained by positron emission tomography), heart period, and high-frequency heart period variability (HF-HPV; an indicator of cardiac parasympathetic activity) were examined in 93 adults (aged 50–70 years) who performed a series of increasingly difficult working-memory tasks. Increased task difficulty resulted in decreased heart period (indicating cardioacceleration) and decreased HF-HPV (indicating decreased cardiac parasympathetic activity). Task-induced decreases in heart period and HF-HPV were associated with concurrent increases and decreases in rCBF to cortical and subcortical brain regions that are speculated to regulate cardiac autonomic activity during behavioral processes: the medial-prefrontal, insular, and anterior cingulate cortices, the amygdala–hippocampal complex, and the cerebellum. These findings replicate and extend a small number of functional neuroimaging studies that suggest an important role for both cortical and subcortical brain systems in human cardiac autonomic regulation.