The development of cholinesterase (ChE) was studied in embryonic rat diaphragm muscles, and aneural and control muscles compared to determine the influence of innervation on its development. (i) ChE deposits developed in their normal positions in aneural muscles, giving a fainter histochemical reaction than in controls. (ii) Muscles denervated at day 16 of gestation, the time at which ChE deposits are first seen normally, and examined at later times had `junctional' deposits with a more normal histological appearance, but there were also many `extra-junctional' deposits scattered across the muscle. (iii) Velocity sedimentation analysis of muscle homogenates revealed that the nerve-specific 16S form of the enzyme was present in aneural muscles. (iv) It is suggested that the special relationship between ACh receptor clusters, ChE deposits and nerve terminals may reflect the presence of an organizing substructure which coordinates their differentiation within developing skeletal muscles. In the absence of the nerve, potential postsynaptic structures begin to develop autonomously.