ABSTRACT The pharyngeal muscles of Caenorhabditis elegans are single sarcomere muscles used for feeding. Like vertebrate cardiac and smooth muscles, C. elegans pharyngeal muscle does not express any of the known members of the MyoD family of myogenic factors. To identify mechanisms regulating gene expression in this tissue, we have characterized a pharyngeal muscle-specific enhancer from myo-2, a myosin heavy chain gene expressed exclusively in pharyngeal muscle. Assaying enhancer function in transgenic animals, we identified three subelements, designated A, B and C, that contribute to myo-2 enhancer activity. These subelements are individually inactive; however, any combination of two or more subelements forms a functional enhancer. The B and C subelements have distinct cell type specificities. A duplication of B activates transcription in a subset of pharyngeal muscles (m3, m4, m5 and m7). A duplication of C activates transcription in all pharyngeal cells, muscle and non-muscle. Thus, the activity of the myo-2 enhancer is regulated by a combination of pharyngeal muscle-type-specific and organ-specific signals. Screening a cDNA expression library, we identified a gene encoding an NK-2 class homeodomain protein, CEH-22, that specifically binds a site necessary for activity of the B subelement. CEH-22 protein is first expressed prior to myogenic differentiation and is present in the same subset of pharyngeal muscles in which B is active. Expression continues throughout embryonic and larval development. This expression pattern suggests CEH-22 plays a key role in pharyngeal muscle-specific activity of the myo-2 enhancer.