Abstract Protein breakdown was monitored in C2C12 myotubes as the rate of release of radioactivity after prelabeling cell protein with [ 3H] tyrosine. IGF-1 (13 nM) and insulin (100 nM) prolonged the half-life of long-lived proteins. Enzymatic activities of cathepsins B and B+L were inhibited by the addition of IGF-1 or insulin. Immunoblotting of cathepsins B and L revealed extensive degradation of heavy chain forms by IGF-1. However, neither expression of cathepsins B and L genes nor expression of cystatin β, an intrinsic inhibitor for cathepsins, was influenced. The addition of E-64, L-3-carboxy-trans-2,3-epoxypropionyl-leucylamide-(4-guanidino) butane, a inhibitor of cathepsins B and L, increased protein contents of heavy chains of cathepsins B and L in the IGF-1 treated cells. Inhibition of protein breakdown by IGF-1 is mediated by autocatalytic inactivation of lysosomal cathepsins B and L.