The time of appearance and the morphological development of cholinesterase activity in the maturing muscle fibers of chick embryos, as related to the innervation, was studied by means of a modified thiolacetic acid method, the acetylthiocholine method, and a protargol silver stain. Four to 21 day embryos, 1, 14 and 28 day ex ovo chickens, and adult birds were studied. In the extrafusal muscle fibers, the following locations of cholinesterase activity were observed: neuromuscular junction, circumjunctional, perijunctional, Z line, diffuse, and myotendinous junction. In the intrafusal muscle fibers, the cholinesterase activity was perinuclear in the equatorial region; in the polar regions, it was at the neuromuscular junctions, in the Z lines, diffuse, and at the myotendinous junctions. Both the junctional and the extrajunctional enzymatic reactions in the extra- and intrafusal fibers were inhibited by M -8 eserine and by M -6 diisopropylfluorophosphate. It was therefore felt that all this activity could be termed "cholinesterase" activity. Prior to innervation, in myoblasts and myoblastic straps, there was abundant cytoplasmic cholinesterase distributed diffusely and in Z lines. After innervation, this activity was gradually lost. From the present studies and from reports in the literature it was concluded that the concentration of cholinesterase activity in certain well limited regions of the extra-and intrafusal fibers (motor end-plates, and possibly the myotendinous junctions and the equatorial regions of intrafusal fibers) only occurred after nerve fibers had established contact with these sites of the muscle fibers. The end-plates formed during embryonic life in the chick embryo were at hatching time well rounded and ring-shaped and were morphologically quite different from the more complicated segmented end-plates of the adult chicken. The question was raised whether the adult end-plate develops from the embryonic ones or whether they are formed ex novo in later stages.