The postnatal development of acetylcholinesterase (AChE, EC 3.1.1.7) and NADH-diaphorase was examined in the caudate-putamen nucleus and substantia nigra of rats ranging from 3 to 90 days in age. From 3 to 15 days post partum islands of AChE and NADH-diaphorase activity were observed in the caudate-putamen nucleus. Individual neuronal somata could also be seen in AChE-stained sections up to 15 days. At later ages neuropil staining became increasingly dense, and this presumably accounted for the infrequent visualization of cell bodies in the brains of older animals. During development AChE appeared in the caudate-putamen nucleus in a lateral to medial topographic order; analogously, enzyme staining in the neostriatum reappeared in the same lateral to medial topographic order in adult rats following irreversible AChE inhibition by intramuscularly injected bis-(1-methylethyl)phosphorofluoridate (di-isopropylfluorophosphate: DFP). Furthermore, DFP treatment in mature animals revealed the presence of AChE in striatal neurons having morphologies similar to those observed in newborn rats. A similar time-course of postnatal AChE development was observed in the substantia nigra. In both the pars compacta and pars reticulata individual cell bodies, which were visible at early ages (3-10 days), became increasingly obscured at later times after birth by extra-somata staining. Between the 6th and 15th postnatal days AChE-containing fibers were seen projecting apparently from pars compacta into pars reticulata. Comparison of the present results with histochemical data of other investigators on the postnatal development of monoamines indicated the likelihood of cholinergicmonoaminergic interactions in the neostriatum and substantia nigra.