There are two main conflicting theories on how the nasolabial crease is formed: a muscular theory and a fascial theory. The muscular theory states that the nasolabial crease is mainly formed by the musculodermal insertions of the lip elevator muscles. The fascial theory claims that the nasolabial crease is mainly formed by dense fibrous tissue and by the firm fascial attachments to the fascia of the lip elevator muscles. If the muscular theory was true, the musculodermal insertions of the facial muscles could be interrupted directly by intradermal injections of low doses of botulinum toxin. Eight cadavers who presented with bilateral nasolabial creases were enrolled in the study. The nasolabial creases were harvested from 14 facial halves in their entire lengths and breadths with 5-mm medial and lateral rims. The horizontally cut samples were stained with hematoxylin-eosin (H&E) and Elastica van Gieson (EVG). Immunohistochemistry for the smooth muscle marker actin and the skeletal muscle marker desmin was also performed. In each of the nasolabial creases, numerous skeletal muscle fibers were found in the dermis, which confirmed the muscular theory of the cause of the nasolabial crease. In addition, muscle fibers were present in the dermis 4 mm medial and 4 mm lateral to the nasolabial crease, but the amounts were significantly less than the amount located directly in the crease. Botulinum toxin injected intradermally into the nasolabial crease might constitute a new treatment option to minimize or even eradicate the crease and the fold. Clin. Anat.2012. © 2012 Wiley Periodicals, Inc.