The development of preferential cholinergic projections of spinal cord neurons within superior cervical ganglia (SCG) was analyzed in vitro using cocultures of SCGs (E17) with organotypic explants of fetal mouse cord (E13). The cord explants consisted of: (1) dorsal vs medioventral strips or mediodorsal vs ventral strips (dissected from levels C8-T4), or (2) transverse sections cut at various levels of the neuraxis. After 4 weeks of coculture, choline acetyltransferase (ChAT) was assayed in individual explants to quantify development of the cholinergic neurotransmitter enzyme (a) within the cord neurons, and (b) within the SCG. An index of cholinergic interaction was calculated as the relative ChAT activity in cocultured ganglion per unit ChAT activity in the ipsilateral cord strip. The highest index value (0.7) was obtained in cocultures with mediodorsal strips of cord. The index of interaction was progressively lower with medioventral (0.4), ventral (0.3) and dorsal (0.1) cord. In cocultures of transverse sections of spinal cord and SCGs, the highest indices of cholinergic interaction (expressed per hemisection of cord) were obtained with cord levels T1/T2 (1.0) and T5 (0.9). The index decreased with T9 (0.7) and was significantly lower with segments C2/C3 (0.3) and L2/L3 (0.19). Addition of a skeletal muscle target explant to the cord-SCG cocultures did not alter the preferential index of interaction between SCG and upper thoracic cord levels. Furthermore, the cholinergic cord neurons in medioventral strips did not promote increase of ChAT activity into equally accessible cocultured ganglia of inappropriate phenotype, e.g. sensory dorsal root ganglia. Decentralization of SCGs after coculture with appropriate T1/T2 cord resulted in loss of ganglionic ChAT activity. Electrical stimulation of the medial region in T1/T2 cord explants evoked compound ganglion action potentials in cocultured SCGs. The ganglion responses were blocked by hexamethonium. These data suggested that neurons located in the medial region of upper thoracic cord (presumably autonomic preganglionic) are able to develop enhanced cholinergic projections within cocultured SCGs, in comparison with neurons located in ventral cord (presumably motoneurons). In contrast, dorsal cord neurons showed no significant cholinergic interaction with SCGs. Furthermore, neurons located in upper thoracic spinal cord segments develop enhanced cholinergic projections within cocultured SCGs in comparison with neurons located in cervical and lumbar cord segments.