The histochemical localization of specific acetylcholinesterase (AcChoEase) and nonspecific cholinesterase (BtChoEase) is described during the early morphogenesis of the whole chicken head with main emphasis on the visual system. It is found that: (i) Expression of AcChoEase is an early differentiation event in the entire brain. Its pattern of first appearance on the external part of the neuroepithelium correlates with the general spatio-temporal pattern of differentiation. AcChoEase thus represents an early differentiation marker. (ii) The late pattern of AcChoEase (at E18), reflecting at least partially the distribution of synaptic AcChoEase shows no direct correlation to the distributions found at early stages when synapses are not yet formed. This argues for a nonsynaptic function of the early appearing AcChoEase. (iii) BtChoEase in early nervous tissue is diffusely localized on the ventricular side of the neural tube. At later stages it becomes concentrated on the ependymal layer as well as along fibers reaching from this inner layer outwards. Minor activities appear in specific external layers of tectum and retina. (iv) During the course of differentiation the enzymes express pronounced graded distributions within the areas where they are detectable. (v) Mesenchymal and epidermal BtChoEase is abundant in the entire head. Prominent amounts of activity are expressed on the rostral epidermis, along the eye cup next to the sclera, and on the rostro-dorsal surrounding of the optic nerves. The results are discussed in the light of possible morphogenetic functions of these enzymes.Buznikov (1) and other investigators (2, 3) suggested possible morphogenetic roles of neurotransmitters and their related protein systems during early embryogenesis -.g., a role for these molecules in addition to synaptic transmission. Evidence from various species suggests very early embryological functions of the cholinergic system (see . In addition to the present search for an embryological and nonsynaptic role of acetylcholinesterase (AcChoEase), there is also increasing interest in attempting to further understand the independence or relationship (or both) of the functions of the enzymes AcChoEase (EC 3.1.1.7) and butyrylcholinesterase (BtChoEase) (EC 3.1.1.8). For the two enzymes, different distributions as well as different sensitivities to eye enucleation were found in the lateral geniculate body and the striate cortex of the adult macaque monkey (11).In the present investigation I have studied the localization* of both enzymes within the whole developing head of the chicken embryo, with main emphasis on the visual system, by using the Karnovsky-Roots technique (12). It is shown how early patterns of AcChoEase and BtChoEase distribution evolve, how they form spatio-temporal concentration gradients and biochemical compartments, and how they interchange into their final patterns (as late as E18) during development. Some general principles derived from these results suggest that cholinesterases may be directly involved in...