PurposeTo describe the anatomic and histologic relations of the muscle of Riolan in the eyelid margin.MethodsSerial microscopic sections of the eyelid were prepared, digitally scanned, and then reconstructed with computer software to create a 3-dimensional profile of this muscle group in two planes.ResultsThe muscle of Riolan is a distinct subdivision of striated muscle that is separate from the pretarsal orbicularis muscle. In parasagittal eyelid sections, the muscle appears to be composed of two separate bundles, the pars ciliaris, located anterior to the tarsal plate, and a second smaller bundle, the pars subtarsalis, located posterior to the orifices of the meibomian glands. Coronal sections, however, demonstrate numerous muscle fibers that traverse the tarsus, connecting the two muscle groups that we describe for the first time as the pars fascicularis.ConclusionsThe three muscle subdivisions are therefore physically joined together and appear to act as a single functional entity that should be collectively referred to as the muscle of Riolan.