A sheep hind-limb preparation used for the study of muscle metabolism by arteriovenous (AV) difference procedures was validated by identifying the muscles which contribute to venous drainage at different positions along the lateral saphenous vein. Dissection of the hind limbs of six mature sheep (three wethers and three ewes) showed that venous blood from the plantar group (M. gastrocnemius, M. soleus, M. plantaris, M. flexo digitorum profundus), and from M. semitendinosus, M. biceps femoris, M. gracilis, M. pectineus and M. adductor muscles entered the lateral saphenous vein but the position of the tip of the blood sampling catheter was found to be critical. In order to sample venous blood from all of the muscles listed above, and to minimize the contribution of blood from non-muscular tissues, blood samples must be taken 25-26 cm from the junction of the cranial and caudal branches of the lateral saphenous vein (for average size sheep of body length about 108 cm and height at withers about 73 cm). The estimation of sheep hind-limb muscle mass is a necessary concomitant of AV difference studies, and a combined tritiated water and dye-dilution procedure has been used to measure both muscle mass and blood flow. The muscle mass estimated in vivo by this technique was closely similar to the true muscle mass obtained by dissection, the range of values of the difference between true and calculated muscle mass expressed as percentage of the true mass being 0.5-16%. It is concluded that these techniques are sufficiently accurate for use in the quantitation of exchange of metabolites across the hind-limb muscle preparation. Patterns of amino acid uptake and release by muscle need to be related to the amino acid profile of the tissue, and the amino acid content of a representative muscle, M. biceps femoris, was determined, and the results compared with published data.