The distribution of acetylcholinesterase activity (acetylcholine acetylhydrolase, EC 220.127.116.11) in the developing human striatum has been studied histochemically in autopsy material from fetal brains of estimated gestational ages 16-29 weeks (180-1000 g) and from the brains of infants 2 days to 4 months old. The findings provide evidence that striatal acetylcholinesterase activity in the human fetus and neonate is concentrated in a network of densely stained zones that appear in cross section as variably shaped 0.5- to 1.0-mm-wide dark patches distributed in a lighter background matrix. An orderly arrangement of the patches seemed well established in the putamen by the 16th-18th week of gestation (crown-rump length 14-15 cm) but in the caudate nucleus the pattern was still not fully elaborated at these ages. The lateroventral part of the caput was mainly dark and its rostromedial margin, though rich early on in pseudocholinesterase activity, was still without strong acetylcholinesterase activity as late as 20-21 weeks (crown-rump length 16-20 cm). The ganglionic eminence at these ages was sharply divided into a dorsal part with little cholinesterase activity and a ventral part with a high content of pseudocholinesterase. Little information was gained about striatal development during late stages of gestation, but in three 5- to 7-month fetal specimens not only dark patches but also patches with dark perimeters and pale centers were present. Clumping of cholinesterase activity appeared at birth and up to the third month of postnatal life. The patches in both caudate nucleus and putamen were dark and of fairly uniform tint in the striatum of the young infant and the matrix staining was darker than in the fetuses. Around the fourth postnatal month hints of the mature pattern were present, with zones of low cholinesterase activity appearing against a dark background in the caudate nucleus and (in one case) a nearly homogeneous staining pattern appearing in the putamen.