Members of the bone morphogenetic protein-1/mammalian tolloid (BMP-1/mTLD) family of proteases cleave diverse extracellular proteins, including the growth inhibitor myostatin. The purpose of this work was to examine the expression of BMP-1/mTLD, tolloid-like-1 and -2 (TLL1 and TLL2) in hindlimb muscles of the mouse in vivo and in C(2)C(12) muscle cells in vitro. Quantitative real-time polymerase chain reaction revealed that neither BMP-1/mTLD nor TLL1 mRNA levels differed between the predominantly fast-twitch tibialis anterior (TA) and gastrocnemius (GAST) muscles and the more slow-twitch soleus (SOL) muscle; TLL2 mRNA levels were not detectable in any of the muscles examined. Interestingly, however, immunohistochemical analysis revealed that BMP-1 protein was expressed in type I and IIa but not in IIb fibers. TLL1 mRNA levels significantly increased in the TA but not the SOL with 3 days of hindlimb suspension and significantly decreased in both TA and SOL in response to 2 days of food deprivation. In contrast, BMP-1/mTLD mRNA levels were unaffected in either muscle by either condition. In addition, BMP-1/mTLD and TLL1 mRNA levels significantly decreased during C(2)C(12) myoblast differentiation in vitro, and activity of a 1,200-bp mouse TLL1 promoter construct was significantly decreased in C(2)C(12) myotubes by differentiation, by mutation of an nuclear factor kappa-beta (NF-kappaB) site, or deletion of a sma/mothers against decapentaplegic (SMAD) site. Together, these data demonstrate that TLL1 mRNA levels are altered by loading, energy status, and differentiation, and thus its expression may be regulated so as to modulate activity of myostatin or other extracellular substrates during these adaptive states.