The right side m. longmissius thoracis et lumborum (LTL) of 18 10-month-old male lambs was injected with the protease inhibitor E-64, and the left side was injected with isotonic saline within 30 min of stunning. Carcasses were hung during chilling by one of three methods; Achilles tendon; pubic symphysis or pubic symphysis with 2-kg weights attached to each hindleg. The LTL section was divided into three portions and aged for either 1, 3 or 7 days. The tenderness, myofibrillar fragmentation and dissociation of extracted actomyosin by pyrophosphate were determined for each portion of LTL. The protease inhibitor increased shear force values by 57%, with a decrease in shear force in the cranial-caudal direction along the LTL. Muscle from Achilles hung carcasses was the toughest. For sarcomere length hanging method had the greatest effect, with muscle from tenderstretched/weighted carcasses having the longest sarcomeres. Injection of an inhibitor caused a significant reduction in myofibrillar fragmentation (P< 0.001) and hanging method and ageing period also significantly affected this characteristic (P< 0.05). Osmolality of samples aged for 1 day was unaffected by any main effect. The amount of actomyosin (mg/g of muscle sample) extracted from muscles injected with the inhibitor was significantly less (P< 0.001) and there was also a significant effect (P< 0.05) of portion on this variable. The relationship between pyrophosphate and the percentage of myosin dissociated from the actomyosin complex was modelled using an exponential function; Y=A - B exp(-kx). Analysis of A, B and k for the 69 samples fitted with an exponential function showed that there was no significant effect (P>0.05) on A and k of any main effect or their interactions. There was, however, a significant effect (P<0.05) of portion on B and also a significant interaction between injection and hanging method (P<0.05). It was found that B did not explain any additional variance in shear force or myofibrillar fragmentation over the main effects and interactions previously found significant. The inhibitor E-64 was effective at preventing tenderisation indicating the role of cysteine proteases in proteolytic degradation. There was no apparent effect of dissociation on tenderness as measured in this experiment and therefore a causal relationship cannot be identified.