We studied possible negative regulators of MyoD using a 27 time point muscle regeneration series in vivo and identified Musculin as a key candidate for transcriptional repression of MyoD. Characterization of Musculin proteins showed two isoforms: the previously characterized 201 aa protein (1a) and a novel 180 aa isoform (1b). We show that the Musculin 1b isoform is equally effective at blocking MyoD-induced transcription as the 1a isoform. Our data narrow the likely repressor domain to between position 158 and 173 in the Musculin protein sequence, and suggest that the induction of Musculin is responsible for the rapid down-regulation of MyoD at 4-5 days during staged regeneration. (c) 2006 Elsevier Inc. All rights reserved.