Development of the peripheral innervation patterns of the L1-S1 lumbosacral ganglia and motor segments in embryonic day 12-17 (E12-17) rat embryos was examined using carbocyanine dyes. Individual dorsal root ganglia (DRGs) and/or isolated ventral horn (VH) segments, or individual peripheral nerves, were isolated in rat embryos fixed at different stages and filled with one of three carbocyanine dyes; DiI, DiA, and DiO. Individual experimental preparations included labeling of 1) single DRGs; 2) multiple DRGs with alternating dyes, DiO, DiI, and DiA; 3) single isolated VH segments; 4) multiple VH segments with alternating dyes; 5) single VH segments and the corresponding segmental DRGs with different dyes; and 6) two or more individual peripheral nerves labeled with different dyes. Results from these preparations have shown that the first fibers exited the lumbar ventral horn and DRGs at E12. At E13 major nerve trunks (e.g., femoral and sciatic) were visible as they exited the plexus region. By E14 afferent fibers were present in the epidermis of the proximal hindlimb, and the major nerve trunks extended into the leg. Fibers originating from L3 to L5 (DRG and VH) reached the paw by E14.5-E15, and the epidermis of the most distal toes was innervated by E16-E16.5. While afferent fibers and motor axons of the same segmental origin mixed extensively in the spinal nerve, fibers of different segmental origin combined in the plexus and major nerve trunks with little or no interfascicular mixing. Dermatomes observed at E14 were in general spotty and non-contiguous. However, by E16 the dermatomes resembled mature forms with substantial overlap only between adjacent ones. Thus the adult pattern of spatial relationships between cutaneous afferent fibers in the periphery is established early in development.