We have identified a negative cis-acting regulatory element in the nicotinic acetylcholine receptor delta-subunit gene's promoter. This element resides within a previously identified 47-base pair activity-dependent enhancer. Proteins that bind this region of DNA were cloned from a lambdagt11 innervated muscle expression library. Two cDNAs (MY1 and MY1a) were isolated that encode members of the Y-box family of transcription factors. MY1/1a RNAs are expressed at relatively high levels in heart, skeletal muscle, testis, glia, and specific regions of the central nervous system. MY1/1a are nuclear proteins that bind specifically to the coding strand of the 47-base pair enhancer and suppress delta-promoter activity in a sequence-specific manner. These results suggest a novel mechanism of repression by MY1/1a, which may contribute to the low level expression of the delta-subunit gene in innervated muscle. Finally, the gene encoding MY1/1a, Yb2, maps to the mid-distal region of mouse chromosome 6.