Background: Fecal incontinence affects people of all ages and social backgrounds and can have devastating psychological and economic consequences. This disorder is largely attributed to decreased mechanical efficiency of the internal anal sphincter (IAS), yet little is known about the pathophysiological mechanisms responsible for the malfunction of sphincteric smooth muscle at the cellular level. Objective: To develop a 3- Dimensional Physiological Model of the IAS Bioengineered in-vitro from Isolated Smooth Muscle Cells. Methods: Smooth muscle cells isolated from the IAS of rabbits were seeded in culture on top of a loose fibrin gel, where they migrated and self-assembled in circumferential alignment. As the cells proliferated, the fibrin gel contracted around a 5mm diameter SYLGARD mold, resulting in a 3-D cylindrical ring of sphincteric tissue. Results: 1) The bioengineered IAS rings generated a spontaneous basal tone. 2) Stimulation with 8-br-cAMP caused a sustained decrease in the basal tone (relaxation), which was calcium-independent. 3) Upon stimulation with acetylcholine, bioengineered IAS rings showed a calcium and concentration-dependent peak contraction at 30 seconds, which was sustained for 4 minutes. 4) Addition of 8-br-cAMP-induced rapid relaxation of acetylcholine-induced contraction and force generation of IAS rings. 5) Lastly, bioengineered sphincter rings show striking functional differences when compared to bioengineered rings made from isolated colonic smooth muscle cells. Conclusions: This is the first report of a 3-D in-vitro model of a gastrointestinal smooth muscle IAS. Bioengineered IAS rings demonstrate physiological functionality and may be used in the elucidation of the mechanisms causing sphincter malfunction.