To identify cortical and subcortical regions involved in voluntary pelvic floor muscle control, functional magnetic resonance imaging (fMRI) was performed at 1.5 T in thirty healthy subjects (15 women, 15 men). The participants performed rhythmical (1 Hz) pelvic floor muscle contractions, which imitated the repetitive interruption of voiding. Since previous reports concerning the representation of pelvic floor muscles in the cortex of the medial wall are inconsistent, a conservative statistical threshold (FWE-corrected P<0.05) was used to detect the most robust foci of activation, and cytoarchitectonic probability maps were used to correlate the results with structural anatomical information. We found a strong and consistent recruitment of the supplementary motor area (SMA), with foci of peak activity located in the posterior portion of the SMA, suggesting that this region is specifically involved in voluntary pelvic floor muscle control. Further significant activations were identified bilaterally in the frontal opercula, the right insular cortex and the right supramarginal gyrus. They may reflect the attentive processing and evaluation of visceral sensations. Weaker signals were detected in the primary motor cortex (M1) and the dorsal pontine tegmentum. There was no significant correlation between bladder volumes and brain activation induced by pelvic floor muscle contractions. We found no significant gender-related differences.